Direct analysis of plasma fibrinogen-derived fibrinopeptides by high-performance liquid chromatography

The fibrinopeptides released from fibrinogen by thrombin can be conveniently quantitated by high-performance liquid chromatography (HPLC). This work describes a method that enables direct analysis of fibrinopeptides derived from fibrinogen contained in small amounts of plasma or whole blood without the necessity of fibrinogen purification. Aliquots of plasma or whole blood were treated with thrombin, diluted in buffer at pH 6.0, boiled and centrifuged. After filtration to 0.22 micron the supernatant was analysed directly by HPLC using a 3 microns C18 analytical column fitted with a pre-column. All the known normal fibrinopeptide peaks and their degradation products were clearly resolved from other plasma-derived peptides and the release pattern was identical to that observed with purified fibrinogen. Some samples contained interfering endogenous peptides which were removed by rapid gel-filtration of 200 microliters of plasma before thrombin treatment. The method was quantitative and the peak heights of fibrinopeptide B (FPB) could be used to accurately measure plasma fibrinogen concentrations directly in samples equivalent to 20 microliters of plasma. These fibrin determinations were unaffected by the type of anticoagulant used were performed on blood samples of both human and animal origin collected and/or stored under a variety of conditions. Variations of the method were specifically developed for different applications requiring quantitative fibrinopeptide analysis.