An assay for anti-factor Xa activity of low molecular weight heparins by high performance liquid size exclusion chromatography

The "gold standard" assay for monitoring low molecular weight heparins (LMWHs) activity is the chromogenic-based anti-factor Xa assay. The methodology of an anti-factor Xa assay is that LMWH is added to a known amount of excess factor Xa and excess antithrombin. It will bind to antithrombin and form a triplet complex with factor Xa, inhibiting the activity of factor Xa. However, the residual factor Xa can still hydrolyze chromogenic peptide substrate, releasing the chromophore for photometric detection. The absorbance is inversely proportional to the amount of heparin/LMWH. The results are given in anticoagulant concentration in units/ mL of anti-factor Xa, such that high values indicate high levels of anticoagulation and low values indicate low levels of anticoagulation. Herein, a novel assay method for anti-FXa activity of LMWHs using high performance liquid size exclusion chromatography (SEC) is reported, in which antithrombin III (AT III) was diluted by the buffer solution contained LMWHs. Subsequently, exogenous FXa and p-nitroaniline coupled peptide substrate were added and incubated for a period, separately. The resulting mixture was separated based on size by SEC, and the free chromophore p-nitroaniline can be detected at an absorption maximum of 385 nm without interference from the absorbance of p-nitroanilide substrates. Moreover, the measurements are not influenced by sample opacity or turbidity, so it is possible to test various complex samples, such as plasma. The assay is robust, sensitive, and cost effective.