USP*效价检测（发色底物法）

Heparin Assays

Anti-Factor IIa Potency

Solutions

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 Heparin Sodium 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 accuray 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 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 °C 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 °C 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 （ΔOD/min）. The blanks for kinetic measurement are also expressed as ΔOD/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 Heparin Sodium in USP Units/mL using statistical methods for parallel-line assays. Express the potency of Heparin Sodium/mg, calculated on the dried basis.

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 Heparin Sodium in USP Units/mL using statistical methods for slope ratio assays. Express the potency of Heparin Sodium/mg, calculated on the dried basis.

Acceptance criteria

The potency of Heparin Sodium, calculated on the dried basis, is NLT 180 USP Heparin Units in each mg.