seminar on assay of oxytocin

1. Presented By
Pradhuman patel
I M.Pharmacy
Dept. of Pharmaceutical Analysis
Submitted to
Dr. C. Shreedhar
Professor and HOD
Dept. of Pharmaceutical Analysis
KARNATAKA COLLEGE OF PHARMACY
Bangalore-560064

2. Oxytocin is a cyclic non-peptide hormone normally produced by the para-
venticular nucleus of the hypothalamus and released by the posterior pituitary
gland.
It is obtained by a process of fractionation from the posterior lobe of the pituitary
gland of healthy oxen or other mammal
It has the property of stimulating contraction of the uterus and milk ejection in
receptive animals.
It may be presented as a solid or as a solution in a solvent containing an
appropriate antimicrobial preservative such as 0.2% w/v of chlorobutanol.
Category:- Oxytocic (facilitating childbirth, especially by stimulating contractions
of the uterus)
NOTE: Fractionation is a separation process in which a certain quantity of a mixture (gas, solid,
liquid, enzymes, suspension, or isotope) is divided during a phase transition, into a number of
smaller quantities (fractions) in which the composition varies according to a gradient.

3. The potency of oxytocin is determined by comparing its activity with that of the
standard preparation of oxytocin under the conditions of a suitable methods of
assay.
STANDARD PREPARATION
The standard preparation is a freeze dried preparation of oxytocin with albumin
and citric acid or any other suitable preparation, the potency of which had been
determined in relation to the International standard.
The Unit is the specific oxytocin activity corresponding to that yielded by 0.0005 g
of the Standard preparation.

4.  Methods :- By Depression of the blood Pressure In chicken
Anaesthetise a young healthy adult chicken weighing (1.2-2.3kg) with a anesthetic
↓
This will maintain a prolonged and constant high B.P
↓
Expose the gluteus primus muscle in one thigh and cut and retract it to reveal the
popliteal artery and crural vein.
↓
Cannulate the popliteal artery and record the blood pressure on a suitable
recorder calibrated for use over a linear range.
↓
Cannulate the crural Vein.

5. Prepare the standard preparation in saline solution so that the volume to be
injected is between 0.1-0.3 ml
↓
Two dose of this solution is injected into cannulated vein, Record the B.P
↓
The injected dose most produce the clearly discriminated, precipitous , sub
minimal decrease in B.P
↓
Normally, the required Dose lies between 20 and 100 milli units.
↓
The time interval between the injection of the must be constant and must lies
between 3 to 10 mins which depends upon the rate at which the B.P returns to
normal.

6. Now dilute the Test preparation with a saline solution so as to obtain the same
response, which is obtained with the standard preparation.
↓
The ratio between the two dose of the Test preparation must be same as that
between the two dose of the standard preparation and this ratio should be
kept constant thoroughly the assay.
The two doses of the Standard preparation and the two doses of the Test
preparation should be given according to a randomized block or a Latin square design
and at least six responses to each should be recorded.
If the animal rapidly becomes insensitive to the repeated injections of the solutions
another animal must be used. Measure all the responses and calculate the result of the
assay by standard statistical methods.

7. HUMAN ANTI HAEMOPHILIC VACCINE

8.  INTRODUCTION:
Human Antihaemophilic vaccine is a preparation of
Antihaemophilic factor which is obtained from human plasma .
It is rich in clotting factor .
PREPRATION OF HUMAN ANTIHAEMOPHILIC VACCINE
The plasma to be used for preparing human antihaemophilic
vaccine is obtained from blood of healthy human donors who
are, as far as can be ascertained after clinical examination ,
laboratory tests on their blood and consideration of their medical
history, free from detectable agents of infection transmissible by
blood transfusion. The examinations and tests to be carried out
are decided by the appropriate national authority.

9. In particular, the blood must be tested with negative results for
a) Evidence of syphilitic infection
b) Hepatitis B surface antigen
c) HIV antibodies by suitably sensitive method.
The haemoglobin value of the donor blood is not less than
12.5%w/v.
The blood is withdrawn aseptically through a closed
system of sterile tubing into a sterile container in which a suitable
anticoagulant solution has been placed before sterilization .
During the withdrawal there is no interruption in the flow from the
donor , and the container is gently agitated .Immediately after the
withdrawal is completed, the blood is cooled to 4°,

10.  If the plasma is to be stored frozen it is separated from the
cellular components by centrifugation and frozen to -30° or
below , preferably within 12 hours of collection ; if the plasma is
not to be frozen it is separated from cellular components by
centrifugation as soon as possible and not later than 18 hours
after collection.

11. CATEGORY: Antihaemophilic to correct deficiencies of coagulation
factor viii.
DESCRIPTION: White or pale yellow powder or friable solid.
STORAGE: Store in an atmosphere of nitrogen in light- resistant
containers at a temperature below 8°.The containers are
sterile and sealed so as to exclude micro-organism.

12. The potency of human antihaemophilic vaccine is determined by
comparing the amount necessary to reduce the clotting time of a
test mixture containing substance that cause clotting of blood with
the amount of the standard preparation necessary to produce the
same effect under the conditions of the following method of assay.

13. STANDARD PREPARATION :
The standard preparation is the 4th international standard for Blood
coagulation factor viii: c, concentrate, human, established in
1989,consisting of an intermediate purity concentrate of human
blood clotting factor viii (supplied in ampoules containing 6.3
units of clotting factor viii), or another suitable preparation the
potency of which has been determined in relation to the
international standard

14. The unit is the specific antihaemophilic factor contained in such a
amount of the standard preparation as the ministry of health and
family welfare ,Govt. of India from time to time indicates as the
quantity exactly equivalent to the unit accepted for international
use.
Special Reagents:
a) Normal serum reagent
b) Phospholipid reagent
c) Clotting factor V solution

15. Dissolve the contents of the sealed container of the substance being
examined in the volume of the liquid state on the label and use
immediately
↓
Reconstitute the entire contents of one ampoule of the standard
preparation as stated on the label and use immediately
↓
To the reconstituted standard preparation and the preparation being
examined, add sufficient imidazole buffer pH 7.4 to produce solution
containing between 0.5 and 2 units per ml.
↓stable for only 15 mins at 20°

16. Use a mixture of 1 volume of a 3.8%w/v solution of sodium citrate
& 5 volumes of saline solution as the diluent
↓
From these solution three successive 2-fold dilutions in the range 1
in 16 to 1 in 256 so that clotting times b/w 17 & 35 secs; dilution
must be accurately made & used immediately
↓
Introduce into six glass tube incubation tubes (75mm * 100mm).
Add 0.1 ml each of special reagents like clotting factor V soln
,phospholipid reagent & normal serum reagent.
↓add 0.1ml of the highest dilution of the
std,prpn(1st tube)

17. Place the tube in water bath at 37°, add 0.1 ml of 0.005M calcium
chloride & start stop watch ,during next minute add 0.1ml of the second
highest dilution of the std to a second tube & place in water bath
↓add 0.05M cacl2(2nd tube) after 1min
Repeat the procedure with the lowest dilution of the std and the highest
to lowest dilution of the prpn being examined so that the cacl2 soln is
added at 2,3,4 and 5mins by the stop watch respectively.
↓
Place in water bath at 37° twelve tubes each containing 0.2ml of 0.025
calcium chloride and a further tube containing about 3 ml of substrate
plasma
↓

18. At 14 mins , 40 secs by the stop watch . Transfer 0.1 ml of the
mixture from the 1st incubation tube to one of the tubes containing
0.2 ml of 0.025 cacl2 soln and mix.
↓
after 15 mins add 0.2ml of the warmed substrate plasma and by
using stop watch record the clotting time interval b/w the addition
of the substrate plasma & 1st indication of fibrin formation .
↓
Repeat the procedure with the incubation tubes at 1min intervals
and carry out a second series of determinations at 21 to 26 mins.
↓

19. Record the clotting time in the corresponding tests and b/w two
series determination don’t differ by more than 5%, showing that
stable plateau of prothrombin activator formation has been
reached .
↓
Carry out a blank determination using in place of the preparation
being examined , an equal quantity of a mixture of 1volume of a
3.8%w/v solution of sodium citrate and 5 volume of the saline soln.
↓
The result of the assay is not valid unless the clotting time in the
blank determination is more than 40 secs .
↓

20. Calculate the result of the assay by standard statistical methods
REFERENCE
IP 1996
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