T/CSBM 0047-2024 Testing method for heparin anti Xa/Ⅱa factor activity and content on medical devices

Scope

Scope: This document specifies the general principles, test methods, and reporting requirements for the determination of heparin anti-Xa/IIa factor activity and content on the surfaces of medical devices coated with heparin anticoagulant. This document applies to heparin-containing coatings integrated into device surfaces by certain methods, and other types of heparin coatings may also be used for reference. Main technical content: 5. Test methods 5.1 Anti-Xa/IIa factor activity 5.1.1 Principle: Heparin forms a complex with AT-III to inhibit the activity of excess added Xa/IIa factors. The chromogenic substrate specifically binds with the remaining Xa/IIa factors, and the released nitrobenzene amine (pNA) in the aqueous solution is measured at 405 nm. The absorbance at 405 nm is inversely proportional to the heparin concentration in the reaction system. 5.1.2 Test procedures 5.1.2.1 Preparation of standard curve: Prepare different concentrations of heparin sodium standard solutions using a pH-8.4 tris-glycine-PEG 6000 buffer solution according to the *Pharmacopoeia of the People's Republic of China* (2020 Edition, Part IV). Add coagulation factor IX to adjust the heparin sodium concentration to 0 IU/mL to 3 IU/mL. Then add Xa/IIa factor solution and chromogenic substrate solution. Incubate at 37 째C for 2 min to 20 min, and terminate the reaction with acetic acid solution. Immediately measure the absorbance at 405 nm of the reaction solution, plot the standard curve with the logarithm of the heparin sodium concentration as the horizontal axis and the absorbance as the vertical axis, and fit the curve using a linear equation. 5.1.2.2 Testing of sample: Place the sample in a test tube and, referring to the steps in 5.1.2.1, input the absorbance value of the sample into the standard curve to calculate the heparin concentration of the sample. 5.1.3 Result analysis Calculate the total heparin anti-Xa/IIa factor activity (Q1) of the sample using Equation (1): ?

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