Determination of the trace levels of urinary fibrinopeptides by high-performance capillary electrophoresis

Objective To establish a high-performance capillary electrophoresis (HPCE)-based method for detection of trace amount of urinary fibrinopeptide A and B (FPA and FPB, respectively) as the specific molecular markers of thrombus formation in vivo. Methods The HPCE system consisted of a 25 cm×50 μm (inner diameter) coated capillary column, 0.1 mol/L phosphoric acid buffer (pH 2.5) and a UV-detector (wavelength at 190 nm). To improve the sensitivity and reproducibility, solid-phase extraction of FPA and FPB in the urine was performed using a Sep-pak C18 column, with a synthetical fibrinopeptide B-Tyr (FPB-Tyr) as the internal standard. Results With this HPCE method, optimal separations of FPA, FPB and FPB-Tyr was achieved within 16 min, with the migration time of 7.28 min, 14.31 min and 15.22 min, respectively. The adjusted peak area ratios of FPA or FPB and the internal standard showed good linearity with the corresponding concentrations of FPA or FPB spiked in the urine(R0.99). Under the above chromatography conditions, the minimum detection concentration of FPA and FPB in untreated urine was 30 μg/L and 40 μg/L, respectively, and the assay precision and recovery of FPA and FPB were acceptable. Conclusion The method we established is reliable and specific for separation and identification of fibrinopeptides and other bioactive peptides.