 COURSE CONTENT

1. BIOLOGICAL ASSAYS

2. ALCOHOL DETERMINATION

3. ALKALOIDAL DRUG ASSAY

4. MISCELLANEOUS DETERMINATION AND TESTS

5. GENERAL KNOWEDGE OF APPENDICES ATTACHED TO B.P.,

B.P.C., AND U.S.P

6. STATISTICAL INTERPRETATION OF QUALITY CONTROL

CHARTS DURING MANUFACTURING PROCESSES

7. QUALITY ASSURANCE OF VACCINES

LEARNING OBJECTIVES:
At the end of this lecture, students will be able to:
 Know about bio assay.
 Define bio assay
 Current trends in bio assay
 Advantages and disadvantages of bio assay.
 When bio assay is conducted
 Preferential method of assay
 Know proper selection , care , handling and other aspects of
animal used for bio assay.
 Know reference standard & why it is used
 Learn assay of different compounds.
 Bioassay is defined as……….
 biological specimens
 Current trend

 Why bioassay is performed????

 Potency/ standardization
 Evaluating suitability of plastic

 Bioassay need is less:

 The majority synthetic
 Less natural
 Disadvantage of bioassay:
 less precise,
 more time-consuming,
 and more expensive to conduct

 Advantage of bioassay
 specificity, sensitivity, or practicality.
 generally are reserved for some use:
1. chemical identity of the active principle has not been
elucidated fully.
2. no adequate chemical assay
3. If the drug is composed of a complex mixture
4. If purification of the crude drug, sufficient for the
performance of a chemical assay, is not possible or
practical.
5. If the chemical assay is not a valid indication of
biological activity.
 Principle of bioassay
 Comparison with international standard preparation
 Reference standard by expert committee of the
biological standardization of WHO
 Potency

 Reduction of biological errors

 ANIMAL TESTING
 Proper selection and adequate care is self-evident.
 Reliable source of animals.
 supply their needs from colonies maintained for this
purpose. E.g specific strain
 For some assays a specific sex must be employed (eg,
estrogenic tests)
 Effect that sex may play in the response should not be
overlooked. (growth rate)
 Animals used in these biological assays should be
handled according to the National Institutes of Health
1. BIOASSAY OF DIGITALIS
 Source of digitalis available
as capsules or tablets.
 Contain glycoside.
 subjected to a biological assay?

PRINCIPLE OF DIGITALIS BIOASSAY

STANDARD PREPARATION AND UNITS:
 The standard preparation is a mixture of dried and
powdered digitalis leaves.
PREPARATION OF EXTRACTS
GUINEA–PIG METHOD:
 Dilution of Standard and test sample
 A guinea pig is anaesthetized & dissected.
 The jugular vein is traced and cannulated.
 The injection is continued through venous cannula until
the heart is arrested in systole.
 Average lethal dose is determined.
 The potency of the test sample is calculated.
PIGEON METHOD:
 Minimum 6 pigeons are used.
 The weight consideration
 Oneside of the wing is dissected and the alar vein is
cannulated by means of a venous cannula.
 Average lethal dose of each sample is determined; The
lethal dose per kg. of body weight is determined for each
pigeon.
 The potency of the test sample is determined.
INSULIN BIOASSAY
 The Rabbit Blood Sugar Method is used
 Diluent preparation
 Standard Stock Solution preparation
 Standard Stock Solution containing 40 USP Insulin Units per
mL
 Standard Solutions preparation
 1.0 USP Insulin Unit per mL (Standard Solution 1)
 2.0 USP Insulin Units per mL (Standard Solution 2).
USP STANDARD SOLUTIONS ASSAY OR TEST SOLUTIONS

1000 IU 1000 IU biological

biological unit in unit in a vial
a vial

Standard Stock Assay/ test Stock

Solution containing Solution containing
40 USP Insulin
40 USP Insulin Units per mL
Units per mL

1.0 USP Insulin 2.0 USP Insulin 2.0 USP Insulin

1.0 USP Insulin
Unit per Units per Unit per mL (test Units per mL (test
mL (Standard mL (Standard Solution 1) Solution 2).
Solution 1) Solution 2).
 Assay Stock Solution preparation
 Assay Solutions—
 1.0 USP Insulin Unit per mL (Assay Solution 1)
 2.0 USP Insulin Units per mL (Assay Solution 2).
 Doses of the Solutions To Be Injected—
 Preparation of Animal—
 Procedure—
 Divide the rabbits into four equal groups of preferably not less
than six rabbits each.
 Inject subcutaneously the doses indicated.
 Second injection being made on the day after the first injection,
or not more than 1 week later.
Group First Injection Second Injection

1 Standard Solution 2 Assay Solution 1

2 Standard Solution 1 Assay Solution 2

3 Assay Solution 2 Standard Solution 1

4 Assay Solution 1 Standard Solution 2

 Blood Sample collection
 Anticoagulant Solution— Dissolve 1 g of edetate sodium
and 200 mg of sodium fluoride in 1 L of water, and mix.
 Dextrose Standard Preparations—
 Test Preparations—
 Procedure—

 Subject the Test Preparations to dialysis across a

semipermeable membrane.
 The absorbances of the Test Preparations are determined at
600 nm in a recording colorimeter.
 VITAMIN D ASSAY(Biological Method)
 The biological assay of vitamin D comprises the recording and
interpretation of observations on groups of rats
 Preliminary Period—
 not longer than 30 days and extends from birth to the first day of
the depletion period
 use a dietary regimen that provides for normal development but
is limited in its content of vitamin D
 Depletion Period—
 extends from the end of the preliminary period to the first day of
the assay period,
 provide each rat ad libitum with the Rachitogenic Diet and water
 Assigning Rats to Groups for Assay Period—
 Consider a litter suitable for the assay period
 The presence of rickets may be established also from Line Test
 Record the weight of each rat
 assign it to a group
 Assay Doses—
 Select two dosage levels of the USP Cholecalciferol RS
 Assay Period—
 extends from the end of the depletion
period for a fixed interval of 7 to 10
days
 cage each rat individually
 On the first and on the third (or
fourth) day of the assay period, feed
each rat one-half of its total assigned
dose.
 dissect out one or more leg bones for
examination by the Line Test.
 Acceptability—
 two-thirds or more but not less than 7 rats show calcification
 If the average score of the standard group on the high dosage
level is not greater than the average score of the standard group
on the low dosage level.
 ANTIBIOTICS—MICROBIAL ASSAYS
 The activity (potency) of antibiotics defined as
 Two general methods are employed for microbial assay:
1. cylinder-plate or “plate” assay
2. turbidimetric or “tube” assay.
 The first depends upon diffusion of the antibiotic through a
solidified agar layer in a petri dish or plate.
 All equipment is to be thoroughly cleaned before and after each
use.
 Temperature Control
 Cylinder-Plate Assay Receptacles
 Turbidimetric Assay Receptacles
 MEDIA PREPARATION
 TEST ORGANISMS
 PREPARATION OF INOCULUM
 MEDIUM 1
 Peptone6.0 gPancreatic Digest of Casein4.0 gYeast Extract3.0 gBeef
Extract1.5 gDextrose1.0 gAgar15.0 gWater1000 mL
 pH after sterilization: 6.6 ± 0.1.
 MEDIUM 2
 Peptone6.0 gYeast Extract3.0 gBeef Extract1.5 gAgar15.0 gWater1000
mL
 pH after sterilization: 6.6 ± 0.1.
 MEDIUM 3
 Peptone5.0 gYeast Extract1.5 gBeef Extract1.5 gSodium Chloride3.5
gDextrose1.0 gDibasic Potassium Phosphate3.68 gMonobasic
Potassium Phosphate1.32 gWater1000 mL
 pH after sterilization: 7.0 ± 0.05.
 Table 2. Test Organisms for Antibiotics Assayed by the
Procedure Indicated in Table 1
 Amikacin Staphylococcus aureus 29737
 Amphotericin B Saccharomycescerevisiae 9763
 Bacitracin Micrococcus luteus 10240
 Bleomycin Mycobacterium smegmatis 607