Chapter-1; Bioassay (Part-3).pptx

“Pharmaceutical Quality Management”
PHR-619
“Bio Assay”
Part-3
By: Dr. Amjad Khan
Assistant Professor, Department of Pharmacy, KUST

Outlines
Assay
Biological Assay (Bio Assay)
Principal of Bio Assay
Techniques for Bio Assay
Topic: Quality Control Charts during Pharmaceutical Manufacturing
Course: Pharmaceutical Quality Management, PHR-619-- Instructor: Dr. Amjad Khan, Assistant. Prof. Department of Pharmacy, KUST -- Email: dr.amjad@kust.edu.pk

Bioassay of Insulin Injection
 Insulin is a hypoglycemic agent i-e it lowers the blood glucose level.
Principle of the Bioassay: The potency of insulin injection (expressed in
International Units/ml) is estimated by comparing the hypoglycemic effect of the
test sample with that of the standard preparation of insulin.
Standard preparation and unit: It is pure, dry and crystalline insulin supplied in
ampoules containing approximately 110-125mg of insulin. 1mg contains 22 Units
of insulin; 1 Unit is equivalent to 45.5 microgram (0.0455 milligram) of insulin.
1mg= 22 Units of Insulin. 1 Unit = 0.0455mg of Insulin.
Topic: Bio Assay

Bioassay of Insulin Injection (Preparation of Standard and Sample Solutions)
Take a specific quantity of insulin and dissolve it in normal saline. Acidify it with HCl
to pH 2.5. Add 0.5% phenol as preservative. Add 1.4% to 1.8% glycerin. Final volume
should contain 20 units/ml. Store the solution in a cool place (2-10 ºC) and use it
within six months.
Preparation of test sample solution: The solution of the test sample is prepared in
the same way as the standard solution described above.
Topic: Bio Assay

Bioassay of Insulin Injection (Suggested Methods)
1. Mouse Convulsion Method
2. Rabbits Blood Sugar Method
Topic: Bio Assay

Bioassay of Insulin Injection (Mouse Convulsion Method)
Principle:
Mice show characteristic hypoglycemic convulsions after S.C. injection of insulin at
elevated temperatures. The percentage convulsions produced by the test and standard
preparations are compared.
Experimental Conditions: Minimum 100 mice weighing between 18-22 grams of
the same strain are used. They should be maintained on a constant and an adequate
diet. They should be fasted 2 hrs prior to the experiment.
Topic: Bio Assay

Mouse Convulsion Method (Standard and Sample Dilution)
Two dilutions of the standard solution are prepared with sterile saline solution
acidified with HCl to pH 2.5, so as to contain 0.030 units/ml (standard dilution-I) and
0.060 units/ml (Standard dilution-II). Similarly, two dilutions of test sample solution
are also prepared.
Dose: Suitable doses of insulin for mice weighing about 20g are 0.015 Units and
0.030 Units. Dilutions of standard and test solutions are prepared in such a way that
the respective doses are contained in equal volumes not greater than 0.5ml.
Topic: Bio Assay

Mouse Convulsion Method (Procedure)
100 Mice are divided into 4 groups each containing 25 mice and insulin is injected
S.C. to each group. A 4-point assay is performed by injecting 2 doses (dilutions) of the
standard solution (S1 & S2) and 2 doses (dilutions) of the test solution (T1 & T2) into
four groups of mice.
Mice are then kept at a constant temperature (29-35°C) in an air incubator with a
transparent font and observed for 1.5 hours following insulin injection. The numbers
of mice which convulse or die are recorded. These mice usually convulse severely but
failure of the animal to upright itself when placed on its back, should as well be
considered as convulsion. Convulsive mice may be saved by an inj. of 0.5 ml. of 5%
dextrose solution. Those animals which survive may be used again for another
experiment after an interval of one week. The death or convulsion in the test animals
are due to the rapid onset of hypoglycemia.
Topic: Bio Assay

Two dilutions of the standard solution are prepared with sterile saline solution
acidified with HCl to pH 2.5, so as to contain 0.030 units/ml (standard dilution-I) and
0.060 units/ml (Standard dilution-II). Similarly, two dilutions of test sample solution
are also prepared.
Dose: Suitable doses of insulin for mice weighing about 20g are 0.015 Units and
0.030 Units. Dilutions of standard and test solutions are prepared in such a way that
the respective doses are contained in equal volumes not greater than 0.5ml.
Topic: Bio Assay

Topic: Bio Assay

Mouse Convulsion Method (Results)
Topic: Bio Assay
Percentage convulsions produced by the test sample are compared with those of the
standard sample. The potency of the test preparation is determined by a specific
statistical method based on the direct proportional relationship between % animals
showing convulsion and the potency of the preparation.

Bioassay of Insulin Injection (Rabbit Blood Sugar Method)
Principle:
Insulin lowers blood sugar level, and the degree of blood sugar lowering is
proportional to the potency of the preparation. The potency of a test sample is
estimated by comparing the hypoglycemic effect (average reduction in the blood
glucose level) of the sample with that of the std. preparation of insulin.
Topic: Bio Assay

Bioassay of Insulin Injection (Rabbit Blood Sugar Method)
Experimental Conditions: 12 healthy rabbits (1.5 to 2 kg) are used. They should then be
maintained on uniform diet but are fasted for 18 hrs before assay.
Standard and sample dilutions: Two dilutions of the standard solution are prepared with
sterile saline solution acidified with HCl to pH 2.5, so as to contain 1 unit/ml (standard
dilution-I) and 2 units/ml (Standard dilution-II). Similarly, two dilutions of test sample
solution are also prepared.
Dose: Suitable doses of insulin for rabbits is about 0.5 units/kg and 1 unit/kg body weight of
the rabbit. Dilutions of standard and test solutions are prepared in such a way that the
respective doses are contained in equal volumes not greater than 1ml.
Topic: Bio Assay

Rabbit Blood Sugar Method (Procedure)
12 Rabbits are divided into 4 groups each containing 3 rabbits. Blood samples are
withdrawn from the marginal ear vein of each rabbit and the average concentration of
blood glucose in mg/dl is determined in each group (initial blood sugar). Then insulin
is injected S.C. to each group. A 4-point assay is performed by injecting 2 doses
(dilutions) of the standard solution (S1 & S2) and 2 doses (dilutions) of the test
solution (T1 & T2) into four groups of rabbits.
Topic: Bio Assay

Topic: Bio Assay

Following insulin injection, blood samples are withdrawn from the marginal ear vein
every hour for 5 consecutive hours and the average concentration of blood glucose in
mg/dl is determined in each group (final blood sugar).
Topic: Bio Assay

Rabbit Blood Sugar Method (Results)
For each dose of the test and standard insulin, the reduction in blood sugar level is
calculated and compared and the potency of the test insulin is determined by a suitable
statistical method.
Topic: Bio Assay

Bioassay of Prepared Digitalis
BIOASSAY OF PREPARED DIGITALIS
Digitalis is a drug that increases the force of myocardial contraction and is used to
treat cardiac failure. It is also known as cardio-tonic as it increases the efficiency of
failure heart. In higher doses it causes stoppage of the heart (cardiac arrest) and death.
Principle of the Bioassay: The Potency of prepared digitalis is estimated by
comparing the action of the test sample of prepared digitalis on the cardiac muscles
with that of the standard preparation of prepared digitalis.
Topic: Bio Assay

Bioassay of Prepared Digitalis
Standard preparation and unit: The standard preparation is a mixture of dried and
powdered digitalis purpurea leaves supplied in vials containing approximately 2.5g of
dried powdered leaves. 1 unit is equivalent to 76 mg; 1mg contains 0.01316 units of
standard preparation of digitalis. 1mg= 0.01316 Units of Digitalis. 1 Unit = 76mg of
Digitalis.
Topic: Bio Assay

Bioassay of Prepared Digitalis (Preparation of Extract)
Preparation of standard extract:
Standard extract of digitalis is prepared by the following procedure:
The contents of a vial containing standard preparation of digitalis are rapidly transferred to a
stoppered weighing bottle and weighed.
The contents are added to a stoppered glass container of at least 50ml capacity.
10ml of alcohol (80%) is added for each g of the powder.
The container is stoppered and is shaken for 24 hrs at 20-30 ºC or for 48hrs at 10-20 ºC.
After shaking the mixture is immediately centrifuged or filtered through a sintered glass filter
with precautions to avoid evaporation of the solvent.
The final extract is transferred to a dry, hard glass bottle which is tightly closed.
The extract is stored at a temperature between 5 ºC and -5 ºC. The extract should be used
within one month.
Topic: Bio Assay

Bioassay of Prepared Digitalis (Preparation of Sample Extract)
Preparation of test sample extract:
The extract of the test sample is prepared in the same way as the standard extract
described above.
Topic: Bio Assay

Bioassay of Prepared Digitalis (Suggested Methods)
1. Guinea–pig Method
2. Pigeon Method
Topic: Bio Assay

Guinea Pig Method (Principle)
Principle: At higher doses digitalis causes stoppage of the heart (cardiac arrest) and
death of guinea pigs, which indicates the end point response of digitalis. The potency
of the test sample is estimated by comparing the lethal dose of the test sample to that
of the standard preparation of digitalis.
Topic: Bio Assay

Guinea Pig Method (Experimental Conditions)
Experimental Conditions: 12 guinea pigs (200-600 g) of the same strain are used.
The weight of the heaviest and the lightest animals should not differ by more than 100
g. Later on when the guinea pigs are divided into 2 groups each containing 6 guinea
pigs, the mean body weights of the two groups should not differ by more than 10 %.
Topic: Bio Assay

Guinea Pig Method (Standard and Sample Dilution)
Standard and sample dilutions: The standard and test sample extracts are diluted
with saline solution until the concentration of alcohol are the same and do not exceed
10 % v/v.
Topic: Bio Assay

Guinea Pig Method (Procedure)
Procedure: 12 guinea pigs are divided in 2 groups each containing 6 guinea pigs. One group is used for
standard preparation and one for the test sample.
The guinea pig is anaesthetized with urethane and the diluted standard extract is injected at a slow and
uniform rate into the vein of a guinea pig. The injection is continued until the heart is arrested (this may
conveniently be determined from an electrical recording). The amount (volume) of extract required to
produce this effect is taken as the lethal dose of the extract. Another set of 5 animals of the same
species are used for this experiment and the average lethal dose (ml per kg of the body weight) of 6
guinea pigs is determined for standard extract. The average lethal dose of the test sample is determined
in a similar way using 6 guinea pigs of the same strain.
Topic: Bio Assay

Guinea Pig Method (Results)
The potency of the test sample is calculated in relation to that of the standard
preparation by dividing the average lethal dose of the sample to the test and expressed
as units per gram.
Topic: Bio Assay

Guinea Pig Method (Results)
Topic: Bio Assay

Bioassay of Prepared Digitalis (Pigeon Method)
2. Pigeon Method:
Principle:
At higher doses digitalis causes stoppage of the heart (cardiac arrest). In pigeons,
stoppage of heart is associated with a characteristic vomiting response called
„Emesis‟. This may be taken as the end point response of digitalis. The potency of the
test sample is estimated by comparing the lethal dose of the test sample to that of the
standard preparation of digitalis.
Topic: Bio Assay

Pigeon Method (Experimental Conditions)
12 pigeons are used. They should be free from gross evidence of disease, obesity or
emaciation (To become extremely thin, especially as a result of starvation or disease).
The weight of the heaviest pigeon should not exceed twice the weight of the lightest
pigeon. Food is withheld 16-28 hours before the experiment. Later on when the
pigeons are divided into 2 groups each containing 6 pigeons, the mean body weights
of the two groups should not differ by more than 30 %.
Topic: Bio Assay

Pigeon Method (Standard and Sample Dilution)
The standard and test sample extracts are diluted with saline solution so that the
estimated lethal dose per kg of the body weight is contained in 15ml.
Topic: Bio Assay

Pigeon Method (Procedure)
12 pigeons are divided in 2 groups each containing 6 pigeons. One group is used for standard preparation and
one for the test sample.
The pigeons are lightly anaesthetized with anesthetic ether and the diluted standard extract is injected
through cannula at a slow and uniform rate into the alar vein of a pigeons. The dose is 1ml per kg of the body
weight and is administered within few seconds & repeated at 5 minutes
interval until the heart is arrested which is associated with emetic response. The amount (volume) of extract
required to produce this effect is taken as the lethal dose of the extract. Another set of 5 pigeons are used for
this experiment and the average lethal dose (ml per kg of the body weight) of 6 pigeons is determined for
standard extract. The average lethal dose of the test sample is determined in a similar way using 6 pigeons.
Topic: Bio Assay

Pigeon Method (Results)
Results: The potency of the test sample is calculated in relation to that of the standard
preparation by dividing the average lethal dose of the sample to the test and expressed
as units per gram.
Topic: Bio Assay

Pigeon Method (Results)
Topic: Bio Assay

Bioassay of Vitamin D
Vitamin D is also known as anti-rachitic vitamin. The deficiency of vitamin D leads to
rickets. Rickets is a disorder caused by a lack of vitamin D, calcium, or phosphate. It
leads to softening and weakening of the bones thus potentially leading to fractures and
deformity. Vitamin D helps the body to control calcium and phosphate levels. If the
blood levels of these minerals become too low, the body may produce hormones that
cause calcium and phosphate to be released from the bones. This leads to weak and
soft bones. The deficiency of Vitamin D is the predominant cause of rickets.
Topic: Bio Assay

Bioassay of Vitamin D (Principle)
The potency of vitamin D is determined by comparing the antirachitic activity of the
test sample of vitamin D with that of the standard preparation of vitamin D. The
antirachitic activity of vitamin D is measured in rats.
Topic: Bio Assay

Bioassay of Vitamin D (Standard Preparation and Unit)
The standard preparation of vitamin D consists of activated crystalline 7-
dehydrocholestrol supplied in bottles as solution in vegetable oils containing 1000
units per gram of the solution. 1 unit is equivalent to 0.000025 mg; 1mg contains
40000 units of pure vitamin D.
1mg= 40000 Units of vitamin D.
1 Unit = 0.000025mg of vitamin D.
Topic: Bio Assay

Bioassay of Vitamin D (Experimental Conditions)
Minimum of 40 young rats of either gender, shortly after being weaned, are used for
the test. About 10 litters (the offspring at one birth of a multiparous mammal- those
which produce more than one at a birth) of 4 rats are selected. (Alternatively 5 litters
of 8 rats can also be selected). 40 rats= 10 litters (groups) of 4 rats (10 ×4 = 40)
Topic: Bio Assay

The weight of the heaviest rat in any litter should not exceed that of the lightest by
more than 10g. All of the 40 rats are fed for about 21 days (3 weeks) on a rachitogenic
diet, which consists mostly of carbohydrates, proteins and electrolytes and no fats.
The development of the necessary degree of rickets may be determined in each rat
under light anaesthesia by taking X-ray photographs of the proximal ends of the tibia
or the distal ends of the ulna and radius.
Topic: Bio Assay

Topic: Bio Assay

Bioassay of Vitamin D (Procedure)
40 rats are divided into 4 groups, each group containing 10 rats.
The groups are made in such a way that one rat from each litter set of 4 or two rats from each
litter set of 8, being assigned to each group. The rats in two groups receive doses of x and nx
units (where n is a suitable value such as 1, 2, 3 or 4), respectively, of standard preparation,
while the rats of the other two groups receive, respectively, doses of test sample preparation in
the same ratio as the doses of the standard preparation (i-e x & nx doses). Suitable doses of
the standard preparation may vary from 2 to 8 units (x=2 units; n=1, or 2, or 3, or 4). Each rat
may receive the whole of its dose at once or the dose may be divided into 8 daily doses.
Topic: Bio Assay

Bioassay of Vitamin D (Procedure)
10-14 days after receiving the doses of standard preparation or of the sample
preparation if given in one amount (single dose), or 10-14 days after receiving the first
fraction of the dose (if given in divided doses), the rats are killed and the extent to
which rickets has been cured is estimated by means of X-ray photographs, or by
examination of the bones after staining, in either instance by reference to a standard
scale.
Topic: Bio Assay

Bioassay of Vitamin D (Results)
The extent to which rickets has been cured by the sample preparation of vitamin D is
compared with that of standard preparation of vitamin D, and the potency of the
sample preparation is calculated by standard statistical methods.
Topic: Bio Assay

References
1. British Pharmacopeia, 2012
2. United States Pharmacopeia (USP/NF), 2018
Topic: Bio Assay

Thank you …
Topic: Bio Assay

Chapter-1; Bioassay (Part-3).pptx

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Chapter-1; Bioassay (Part-3).pptx

Similar to Chapter-1; Bioassay (Part-3).pptx (20)

Recently uploaded

Recently uploaded (20)

Chapter-1; Bioassay (Part-3).pptx