This document summarizes several bioassay methods for estimating the potency of adrenaline (epinephrine) samples, including methods using anesthetized dogs, spinal cats, rabbit duodenums, rat uteruses, and isolated rabbit hearts. The potency of a test adrenaline sample is estimated by comparing its physiological effects, such as increases in blood pressure or force of heart contraction, to those of a standard adrenaline preparation with a known potency. The document describes the experimental procedures and physiological readouts for each bioassay method.

1. BIOASSAY
OF
ADRENALINE
KOPPALA RVS CHAITANYA
ASSISTANT PROFESSOR
DEPARTMENT OF PHARMACOLOY

2. PRINCIPLE: Potency of test sample is estimated by comparing the rise in BP of the test sample with
the produced by the standard preparation of adrenaline.
STANDARD PREPARATION: It should fulfill all the tests of purity as per adrenaline
monograph is the Indian Pharmacopoeia. The specific rotation of 4%w/v solution of the standard adrenaline
in 0.1 N HCl should be -5°C to 5°C dilution of adrenaline is prepared by dilution with saline. Suitable
concentration 1:10000. The test sample is also prepared with saline.
METHOD USING ANAESTHETISED DOG:
➢ A medium sized dog (10-13 Kgs) is anaesthetized with phenobarbitone sodium in a dose of 25mg/kg
I.V. artificial respiration is given through tracheal tube if necessary. BP is recorded in a kymograph
after cannulating carotid artery with arterial cannula and same with manometer.
➢ The animal should be given 0.001-0.002mg of atropine sulphate to paralyze the vagi. This can be
confirmed by electrical stimulation of vagus. There will be no fall in BP in atropinized animal after
electrical stimulation.
➢ Adrenaline is injected into femoral vein through venous cannula. Two successive responses of the same
dose of adrenaline are noted. The similar responses after the same dose of adrenaline indication no
variation in BP and animal is ready for the assay. Then the alternative test and standard samples are
given till both produce similar rise in BP. Injection given at 5min time interval. The potency of test
sample is compared with that of standard sample.
ON SPINAL CAT: Similar to the pressor activity of anaesthetized dog.
METHOD USING RABBIT’S DUODENUM:
➢ A rabbit weighing 2-3 kgs is killed by head blow method and bled to death. The animal is dissected out
and duodenum is isolated.
➢ It is suspended in the inner bath of mammalian organ bath. Inner bath is filled with Tyrode solution
maintained at 37.5 °C.
➢ The tissue is allowed to stabilize for 30min. Rhythmic contractions are recorded by isotonic frontal
lever on kymograph.
➢ Adrenaline relaxes the duodenum and the same property taken up to consideration for finding out the
potency of the test sample. It is bio assayed by graphical method.
DE JALON’S METHOD ON RAT UTERUS:

3. ➢ First demonstrated by De Jalon a virgin female rat, weighing 100-150g is used. The rat is killed by head
blow method.
➢ the uterine horns are isolated and placed in a petri dish containing de Jalon solution. Uterine horn is
ligated and suspended in a mammalian organ bath containing modified Ringer solution having the
composition. NaCl -9.0g; CaCl2- 0.06mg; KCl-0.45g; dextrose- 0.5g; NaHCO3-0.5g and distilled
water -1000ml. This solution abolishes the rhythmic contraction of the uterus, maintained at 37°c and
bubbled with air.
➢ Two identical contractions for 30sec with carbachol are recorded (0.75mcg/ml). Then selecting three
different doses of std adrenaline and one intermediate dose test adrenaline. The % reduction of
carbachol contractions are recorded.
STRAUB’S METHOD:
➢ A glass cannula is passed through the aorta and valves into the ventricle of the heart so that the solution
is pumped up and down in the cannula with every beat.
➢ A graph paper attached behind the cannula to give an estimate of the force of contraction of systole of
the heart at every beat.
➢ The beat is recorded by attaching the tip of the ventricle through a thread to a lever. This preparation
widely used for the detection of small quantities of adrenalin.
➢ Adrenaline increases the force of contraction of ventricle. The increased force of contraction produced
by the test sample is compared with that of the standard preparation.