Anti-factor Xa activity—
pH 8.4 Buffer—
Dissolve amounts of tris(hydroxymethyl)aminomethane, edetic acid, and sodium chloride in water containing 0.1% of polyethylene glycol 6000 to obtain a solution having concentrations of 0.050 M, 0.0075 M, and 0.175 M, respectively. Adjust, if necessary, with hydrochloric acid or sodium hydroxide solution to a pH of 8.4.
Antithrombin III solution—
Reconstitute an accurately weighed quantity of antithrombin III (see Reagent Specifications under Reagents, Indicators, and Solutions) in pH 8.4 Buffer to obtain a solution having a concentration of 1.0 Antithrombin III Unit per mL.
Factor Xa solution—
Reconstitute an accurately weighed quantity of bovine factor X
a (see Factor X
a in
Reagent Specifications under
Reagents, Indicators, and Solutions) in
pH 8.4 Buffer to obtain a solution that gives an absorbance value between 0.65 and 1.25 at 405 nm when assayed as described below but using 30 µL of
pH 8.4 Buffer instead of 30 µL of the
Standard solutions or the
Test solutions.
Note—Factor Xa solution contains about 3 nanokatalytic units per mL, but can vary depending upon the manufacturer of factor Xa or the substrate used.
Chromogenic substrate solution—
Prepare a solution of a suitable chromogenic substrate for amidolytic test (see Reagent Specifications under Reagents, Indicators, and Solutions) specific for factor Xa in water to obtain a concentration of about 1 mM.
Stopping solution—
Prepare a 20% (v/v) solution of acetic acid in water.
Standard solutions—
Dilute an accurately measured volume of
USP Heparin Sodium RS with
pH 8.4 Buffer to obtain at least 5 (out of 7 below) solutions having known activities of about 0.375, 0.3125, 0.25, 0.188, 0.125, 0.0625, and 0.0313 USP Heparin Unit per mL.
Test solutions—
Dissolve or dilute an accurately measured quantity of Heparin Sodium in pH 8.4 Buffer, and dilute with the same buffer to obtain solutions having activities approximately equal to those of the Standard solutions.
Procedure—
[Note—Perform the test with each
Standard solution and
Test solution in duplicate.
] To each of a series of suitable plastic tubes placed in a water bath set at 37
, transfer 120 µL of
pH 8.4 Buffer. Then separately transfer 30 µL of the different dilutions of the
Standard solutions or the
Test solutions to the tubes. Add 150 µL of
Antithrombin III solution, prewarmed at 37
for 15 minutes, to each tube, mix, and incubate for 2 minutes. Add 300 µL of
Factor Xa solution, prewarmed at 37
for 15 minutes, to each tube, mix, and incubate for 2 minutes. Add 300 µL of
Chromogenic substrate solution, prewarmed at 37
for 15 minutes, to each tube, mix, and incubate for exactly 2 minutes. Add 150 µL of
Stopping solution to each tube, and mix. Prepare a blank for zeroing the spectrophotometer by adding the reagents in reverse order, starting with the
Stopping solution and ending with the addition of 150 µL of
pH 8.4 Buffer, and excluding the
Standard solutions or the
Test solutions. Record the absorbance at 405 nm against the blank.
Calculations—
Plot the log of the absorbance values of the
Standard solutions and the
Test solutions versus heparin concentrations in USP Units. Construct separate straight lines of best fit using least-squares linear regression analyses for the
Standard solutions and the
Test solutions, and determine the slope for each regression line. Calculate the potency of Heparin Sodium by the formula:
P(ST / SS),
in which
P is the potency of
USP Heparin Sodium RS; and
ST and
SS are the slopes of the lines from the
Test solutions and the
Standard solutions, respectively. Express the Anti-factor X
a potency of the
Test solution as a percentage of the heparin concentration determined in the
Assay. Calculate the percentage of anti-factor X
a activity against anticoagulant activity by the formula:
100(anti-factor Xa potency / anticoagulant potency).
Not less than 80% and not more than 120% is found.
Assay—
Standard preparation—
Determine by preliminary trial, if necessary, approximately the minimum quantity of
USP Heparin Sodium RS which, when added in 0.8 mL of saline TS, maintains fluidity in 1 mL of prepared plasma for 1 hour after the addition of 0.2 mL of calcium chloride solution (1 in 100). This quantity is usually between 1 and 3 USP Heparin Units. On the day of the assay prepare a
Standard preparation such that it contains, in each 0.8 mL of
saline TS, the above-determined quantity of the Reference Standard.
Assay preparation—
Dissolve about 25 mg of Heparin Sodium, accurately weighed, in sufficient saline TS to give a concentration of 1 mg per mL, and dilute quantitatively to a concentration estimated to correspond to that of the Standard preparation.
Preparation of plasma—
Collect blood from sheep directly into a vessel containing 8% sodium citrate solution in the proportion of one volume to each 19 volumes of blood to be collected. Mix immediately by gentle agitation and inversion of the vessel. Promptly centrifuge the blood, and pool the separated plasma. To a 1-mL portion of the pooled plasma in a clean test tube add 0.2 mL of calcium chloride solution (1 in 100), and mix. Consider the plasma suitable for use if a solid clot forms within 5 minutes. To store plasma for future use, subdivide the pooled lot into portions not exceeding 100 mL in volume, and store in the frozen state, preventing even partial thawing prior to use. For use in the assay, thaw the frozen plasma in a water bath at a temperature not exceeding 37
. Remove particulate matter by straining the thawed plasma through a coarse filter.
Procedure—
To meticulously clean 13-mm × 100-mm test tubes add graded amounts of the
Standard preparation, selecting the amounts so that the largest 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 so prepared add sufficient saline TS to make the total volume 0.8 mL. Add 1.0 mL of prepared plasma to each tube. Then add 0.2 mL of calcium chloride solution (1 in 100), note the time, immediately insert a suitable stopper in each tube, 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 Assay preparation, completing the entire process of preparing and mixing the tubes of both the Standard preparation and the Assay preparation within 20 minutes after the addition of the prepared plasma. One hour, accurately timed, after the addition of the calcium chloride, determine the extent of clotting in each tube, recognizing three grades (0.25, 0.50, and 0.75) between zero and full clotting (1.0). If the series does not contain 2 tubes graded more than 0.5 and 2 tubes graded less than 0.5, repeat the assay, using appropriately modified Standard preparation and Assay preparation.
Calculation—
Convert to logarithms the volumes of
Standard preparation used in the successive 5 or 6 tubes that bracket a grade of clotting of 0.5, including at least 2 tubes with a larger and 2 tubes with a smaller grade than 0.5. Number and list the tubes serially, and tabulate for each the grade of clotting observed in each tube. From the log-volumes,
x, and separately from their corresponding grades of clotting,
y, compute the paired averages
xi and
yi of Tubes 1, 2, and 3, of Tubes 2, 3, and 4, of Tubes 3, 4, and 5, and, where the series consists of 6 tubes, of Tubes 4, 5, and 6, respectively. If for one of these paired averages the average grade,
yi, is exactly 0.50, the corresponding
xi is the median log-volume of the
Standard preparation, xS. Otherwise, interpolate
xS from the paired values of
yi,
xi and
yi +1,
xi +1 that fall immediately below and above grade 0.5 as
xS =
xi + (
yi 
0.5)(
xi +1 xi) / (
yi yi +1).
From the paired data on the tubes of the
Assay preparation, compute similarly its median log-volume
xU.
The log potency of the Assay preparation is
M = xS xU + log
R,
where R = vS /vU is the ratio of the USP Heparin Units (vS) per mL of the Standard preparation to the mg (vU) of Heparin Sodium per mL of the Assay preparation.
Repeat the assay independently, and average the two or more values of
M to obtain
bar(M). If the second determination of
M differs by more than 0.05 from the first determination, continue the assay until the log confidence interval computed as directed under
Confidence Intervals for Individual Assays in
Design and Analysis of Biological Assays 
111
does not exceed 0.20. The potency of Heparin Sodium in USP Heparin Units per mg is P
* = antilog
bar(M).
