Saturday 12 May 2012

Biological Assay of Heparin Sodium

Biological Assay of Heparin Sodium

Principle: The potency of heparin sodium is determined by comparing the concentration
necessary to prevent the clotting of sheep or goat or human plasma with the concentration
of the Standard Preparation of heparin sodium necessary to give the same effect under the
conditions of the following method of assay.


Standard Preparation and Unit: The Standard Preparation is the freeze-dried sodium salt
of the purified active principle from bovine  intestinal mucous membranes or any other
suitable preparation, the potency of which has been determined in relation to the
International Standard. The Unit is the specific activity contained in 7.7 ยต g of the
Standard Preparation and is the same as the International Unit; 1 mg contains 130 Units.

Special Reagents: Prepared Plasma: Collect blood from sheep or goats or human
volunteers directly into a vessel containing 8% w/v solution of  sodium  citrate in the
proportion of 1 volume to each 19 volumes of blood to be collected. Mix immediately by
gentle agitation and inversion of the vessel. Immediately centrifuge and pool the
separated plasma. To 1 ml of the pooled plasma in a clean test-tube add 0.2 ml of a 1%
w/v solution of  calcium  chloride and mix. The plasma is suitable if a solid clot forms
within 5 minutes.

Solution of standard preparation: Determine by preliminary trial, if necessary,
approximately the minimum quantity of the Standard Preparation of heparin sodium
which, when added in 0.8 ml of saline solution, maintains fluidity in 1 ml of prepared
plasma for 1 hour after the addition of 0.2 ml of a 1% w/v solution of calcium chloride.
On the day of the assay prepare a solution of the Standard Preparation such that it
contains in each 0.8 ml of saline solution the above-determined quantity of the Standard
Preparation.

Test solution: Weigh accurately about 25 mg of the preparation being examined and
dissolve in sufficient saline solution to give a concentration of 1 mg per ml and dilute to a
concentration estimated to correspond to that of the solution of the Standard Preparation.

Method: To very clean test-tubes (150 mm x 16 mm) add graded amounts of the solution
of standard preparation, selecting the amounts so that the largest dose does not exceed
0.8 ml and so that they correspond roughly to a geometric series in which each step is
approximately 5% greater than the next lower. To each tube add sufficient saline solution
to make the total volume 0.8 ml. Add 1.0 ml of prepared plasma to each tube. Then add
  170.2 ml of a 1% w/v solution of calcium chloride, note the time, immediately stopper each
tube with a suitable stopper and mix the contents by inverting three times in such a way
that the entire inner surface of the tube is wet.
In the same manner set up a series using the test solution, completing the entire process
of preparing and mixing the tubes of both the  solution of  standard preparation and the
test  solution within 20 minutes after the addition of the  prepared  plasma. Exactly one
hour after the addition of the calcium chloride solution, determine the extent of clotting in
each tube, recognizing three grades between zero and full clotting.
The dilution of the test solution which contains heparin sodium in the same concentration
as the dilution of the  solution of  standard preparation is that contained in the series of
dilutions which show the same degree of clotting as the series of dilutions of the solution
of standard preparation. If the degree of clotting observed in the series of dilutions of the
solution  of  standard  preparation lies between that observed in two of the series of
dilutions of the sample being examined, the potency of the latter is estimated. If there is
no such correspondence between the degrees of clotting produced by the  solution  of
standard  preparation and any of the dilutions of the sample being examined, new
dilutions of the latter are prepared and assay is repeated.
Carry out no fewer than three independent assays. Calculate the estimated potency of the
preparation being examined by combining the results of these assays by standard
statistical methods.
Limits of error - The limits of error (P = 0.99) attainable with the test are:
90 and 110%, with three determinations;
92 and 108%, with four determinations.
Heparin Sodium intended for use in the manufacture of injectable preparations without a
further procedure for the removal of pyrogens complies with the following additional
requirement.

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