Anticoagulant Heparin Solution
DEFINITION
Anticoagulant Heparin Solution is a sterile solution of Heparin Sodium in Sodium Chloride Injection. Its
potency is NLT 90.0% and NMT 110.0% of the potency stated on the label in terms of USP Heparin Units.
It contains NLT 0.85% and NMT 0.95% of sodium chloride (NaCl). It may be buffered. It contains no
antimicrobial agents.
Prepare Anticoagulant Heparin Solution as follows.
Add the Heparin Sodium, in solid form or in solution, to the Sodium Chloride Injection, mix, filter if
necessary, and sterilize.
ASSAY
• ANTI-FACTOR IIA POTENCY
pH 8.4 buffer: Dissolve 6.10 g of tris(hydroxymethyl)aminomethane, 10.20 g of sodium chloride, 2.80 g
of edetate sodium, and, if suitable, between 0 and 10.00 g of polyethylene glycol 6000 and/or 2.00 g
of bovine serum albumin in 800 mL of water. [NOTE—2.00 g of human albumin may be substituted for
2.00 g of bovine serum albumin. ] Adjust with hydrochloric acid to a pH of 8.4, and dilute with water to
1000 mL.
Antithrombin solution: Reconstitute a vial of antithrombin (see Reagents, Indicators, and Solutions—
Reagent Specifications) in water to obtain a solution of 5 Antithrombin IU/mL. Dilute this solution with
pH 8.4 buffer to obtain a solution having a concentration of 0.125 Antithrombin IU/mL.
Thrombin human solution: Reconstitute thrombin human (factor IIa) (see Reagents, Indicators, and
Solutions—Reagent Specifications) in water to give 20 Thrombin IU/mL, and dilute with pH 8.4 buffer
to obtain a solution having a concentration of 5 Thrombin IU/mL. [NOTE—The thrombin should have a
specific activity of NLT 750 IU/mg. ]
Chromogenic substrate solution: Prepare a solution of a suitable chromogenic thrombin substrate for
amidolytic test (see Reagents, Indicators, and Solutions—Reagent Specifications) in water to obtain a
concentration of 1.25 mM.
Stopping solution: 20% (v/v) solution of acetic acid
Standard solutions: Reconstitute the entire contents of an ampule of USP Heparin Sodium for Assays
RS with water, and dilute with pH 8.4 buffer to obtain at least four dilutions in the concentration range
between 0.005 and 0.03 USP Heparin Unit/mL.
Sample solutions: Proceed as directed for Standard solutions to obtain concentrations of
Anticoagulant Heparin Solution similar to those obtained for the Standard solutions.
Analysis
[NOTE—The procedure can also be performed using alternative platforms. ]
For each dilution of the Standard solutions and the Sample solutions, at least duplicate samples
should be tested. Label a suitable number of tubes, depending on the number of replicates to be
tested. For example, if five blanks are to be used: B1, B2, B3, B4, and B5 for the blanks; T1, T2, T3,
and T4 each at least in duplicate for the dilutions of the Sample solutions; and S1, S2, S3, and S4
each at least in duplicate for the dilutions of the Standard solutions. Distribute the blanks over the
series in such a way that they accurately represent the behavior of the reagents during the
experiments. [NOTE—Treat the tubes in the order B1, S1, S2, S3, S4, B2, T1, T2, T3, T4, B3, T1,
T2, T3, T4, B4, S1, S2, S3, S4, B5. ] Note that after each addition of a reagent, the incubation
Heparin Sodium 75,000 Units
Sodium Chloride Injection,
sufficient quantity to make 1000 mL
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mixture should be mixed without allowing bubbles to form. Add twice the volume (100–200 µL) of
Antithrombin solution to each tube containing one volume (50–100 µL) of either the pH 8.4 buffer or
an appropriate dilution of the Standard solutions or the Sample solutions. Mix, but do not allow
bubbles to form. Incubate at 37 for at least 1 min. Add to each tube 25–50 µL of Thrombin human
solution, and incubate for at least 1 min. Add 50–100 µL of Chromogenic substrate solution. Please
note that all reagents, Standard solutions, and Sample solutions should be prewarmed to 37 just
before use. Two different types of measurements can be recorded:
1. Endpoint measurement: Stop the reaction after at least 1 min with 50–100 µL of Stopping
solution. Measure the absorbance of each solution at 405 nm using a suitable
spectrophotometer (see Spectrophotometry and Light-Scattering 851 ). The RSD over the
blank readings is less than 10%.
2. Kinetic measurement: Follow the change in absorbance for each solution over 1 min at 405 nm
using a suitable spectrophotometer (see Spectrophotometry and Light-Scattering 851 ).
Calculate the change in absorbance/min (OD/min). The blanks for kinetic measurement are
also expressed as OD/min and should give the highest values because they are carried out
in the absence of heparin. The RSD over the blank readings is less than 10%.
Calculations: The statistical models for Slope ratio assay or Parallel-line assay can be used,
depending on which model best describes the correlation between concentration and response.
Parallel-line assay: For each series, calculate the regression of the absorbance or change in
absorbance/min against log concentrations of the Standard solutions and the Sample solutions, and
calculate the potency of Anticoagulant Heparin Solution in USP Units/mL using statistical methods for
parallel-line assays.
Slope ratio assay: For each series, calculate the regression of the log absorbance or the log change in
absorbance/min against concentrations of the Standard solutions and the Sample solutions, and
calculate the potency of Anticoagulant Heparin Solution in USP Units/mL using statistical methods for
slope ratio assays.
Acceptance criteria: 90.0%–110.0% of the potency stated on the label in terms of USP Heparin Units.
• SODIUM CHLORIDE
Sample solution: Solution and potassium chromate TS (5:1)
Analysis: Titrate with 0.1 N silver nitrate VS. Each mL of 0.1 N silver nitrate is equivalent to 5.844 mg
of NaCl.
SPECIFIC TESTS
• PH 791 : Between 5.0 and 7.5
• BACTERIAL ENDOTOXINS 85 : It contains NMT 2.5 USP Endotoxin Units/mL.
• INJECTIONS 1 : Meets the requirements
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in single-dose containers, of colorless, transparent Type I or Type
II glass, or of a suitable plastic material (see Transfusion and Infusion Assemblies and Similar Medical
Devices 161 ).
• LABELING: Label it in terms of USP Heparin Units, and to indicate the number of mL of Solution required
per 100 mL of whole blood.
• USP REFERENCE STANDARDS 11
USP ENDOTOXIN RS
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USP HEPARIN SODIUM FOR ASSAYS RS
Auxiliary Information— Please check for your question in the FAQs before contacting USP.
USP34–NF29 Page 3040
Topic/Question Contact Expert Committee
Monograph Anita Y. Szajek, Ph.D.
Principal Scientific Liaison
1-301-816-8325
(BIO12010) Monographs - Biologics and
Biotechnology 1
85 Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison
1-301-816-8339
(GCM2010) General Chapters - Microbiology
Reference
Standards
RS Technical Services
1-301-816-8129
rstech@usp.org
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