高效毛细管电泳法检测尿液中痕量纤维蛋白肽A、B

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

目的建立高效毛细管电泳测定尿液中反应体内微血栓形成的特异分子标志物纤维蛋白肽A(FPA)和纤维蛋白肽B(FPB)的方法。方法采用25cm×50μm涂层毛细管柱,0.1mol/LpH2.5的磷酸缓冲液,27.58kPa压力进样,190nm紫外检测。用比FPB多一个酪氨酸的合成肽(FPB-Tyr)作内标,加内标后的尿样用Sep-PakC18柱预处理。结果采用本法,尿样中的FPA、FPB和内标可在16min内得到很好的分离,三者的迁移时间分别为:7.28、14.31、15.22min;将内标和一系列浓度的FPA、FPB标准品加入空白尿样,用Sep-PakC18柱预处理后,毛细管电泳进样分析,在FPA和FPB浓度为0～40mg/L的范围内,用FPA(FPB)同内标的校正峰面积比值对添加的相应的FPA(FPB)浓度得到校正工作曲线,线性关系良好,R均>0.99。未预处理的尿样中FPA、FPB的最低检出浓度分别为30μg/L、40μg/L(信噪比为3∶1);本法FPA和FPB测定的日内、日间精密度好,平均回收率高。结论本方法可靠,特异性好,可为开展纤维蛋白肽类及其它的痕量肽类分析提供参考。

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 16min, withthe 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（R〉0.99）. 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.