Vincenzi Jager Alessandra, Franco Maggi Tavares Marina
Instituto de Química, Universidade de São Paulo, Av Prof Lineu Prestes 748, CP 26077, 05513-970, São Paulo, SP, Brazil.
J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Mar 5;785(2):285-92. doi: 10.1016/s1570-0232(02)00917-0.
In this work, a novel CIEF methodology for the analysis of the glycated hemoglobin, HbA(1c), in dimethylpolysiloxane coated fused-silica capillaries (DB-1, 50 microm I.D., 27 cm, 0.20 microm coating thickness), using a narrow pH ampholyte mixture (4% pH 6-8:pH 3-10, 10:1, v/v) in 0.30% methylcellulose, was developed. In the focusing procedure, a 0.100-mol l(-1) phosphoric acid solution was used as anolyte and a 0.040-mol l(-1) NaOH solution was used as catholyte. During method development, two types of mobilization of the focused hemoglobins were tested: pressure and chemical mobilization. Chemical mobilization performed better, allowing the complete baseline resolution of the hemoglobin of interest, HbA(1c), from its adjacent peak, HbA, in less than 8 min. In the chemical mobilization procedure, the catholyte was replaced by a 0.040-mol l(-1) NaOH solution containing 0.080 mol l(-1) NaCl. The proposed methodology was applied to the analysis of 31 hemolysate samples and validated with respect to the selectivity, inter-assay and intra-assay precision (both migration time and hemoglobin percentage concentration). In addition, HbA(1c) determinations were compared for the CIEF method and a chromatographic standardized procedure using cation-exchanger columns (Variant, Bio-Rad), adopted in a local clinical laboratory, showing excellent correlation (r(2)=0.872, n=31). The slope was found to be statistically equal to one but the intercept differed from zero. Also the Bland-Altman plot indicates bias, implying that the CIEF method yields HbA(1c) concentration higher than the reference method. The separation of the hemoglobins HbA, HbA(2), HbF and HbA(1c) and the variants HbS and HbC was also demonstrated (8 min run). The resolving power of the proposed CIEF method allowed baseline resolution of hemoglobins with a pI difference as small as ca. 0.03, as it is the case for the pairs HbC/HbA(2) and HbA/HbA(1c).
在本研究中，开发了一种新型毛细管等速电泳（CIEF）方法，用于分析涂覆二甲基聚硅氧烷的熔融石英毛细管（DB-1，内径50μm，长27cm，涂层厚度0.20μm）中的糖化血红蛋白HbA(1c)，使用0.30%甲基纤维素中的窄pH范围两性电解质混合物（4% pH 6 - 8:pH 3 - 10，体积比10:1）。在聚焦过程中，使用0.100 mol/L磷酸溶液作为阳极电解液，0.040 mol/L氢氧化钠溶液作为阴极电解液。在方法开发过程中，测试了两种聚焦血红蛋白的迁移方式：压力迁移和化学迁移。化学迁移效果更好，能够在不到8分钟的时间内使目标血红蛋白HbA(1c)与其相邻峰HbA实现完全基线分离。在化学迁移过程中，阴极电解液被含有0.080 mol/L氯化钠的0.040 mol/L氢氧化钠溶液取代。将所提出的方法应用于31份溶血样品的分析，并在选择性、批间和批内精密度（迁移时间和血红蛋白百分比浓度）方面进行了验证。此外，将CIEF方法测定的HbA(1c)结果与当地临床实验室采用的使用阳离子交换柱（Variant，Bio-Rad）的色谱标准化程序进行了比较，显示出极好的相关性（r(2)=0.872，n = 31）。发现斜率在统计学上等于1，但截距不为零。布兰德 - 奥特曼图也表明存在偏差，这意味着CIEF方法得出的HbA(1c)浓度高于参考方法。还展示了血红蛋白HbA、HbA(2) HbF和HbA(1c)以及变体HbS和HbC的分离（运行8分钟）。所提出的CIEF方法的分离能力能够实现pI差异小至约0.03的血红蛋白的基线分离，如HbC/HbA(2)和HbA/HbA(1c)对的情况。
