Lauff J J, Kasper M E, Ambrose R T
J Chromatogr. 1981 Dec 11;226(2):391-402. doi: 10.1016/s0378-4347(00)86073-9.
A high-performance, reversed-phase liquid chromatographic (HPLC) procedure has been developed for the separation of at least three major bilirubin fractions in bile and four fractions in human serum. This procedure was unlike most others, in that serum was not totally deproteinized prior to injection onto the HPLC column; instead, serum was treated with an excess of sodium sulfate solution to precipitate primarily proteins larger than albumin. Injection of the filtered and diluted supernatant onto a reversed-phase column then resulted in the separation of the bilirubin species in a 24-min gradient elution run. Both the initial aqueous acidic mobile phase and the final isopropyl alcohol-based mobile phase contained 5% methoxyethanol (v/v) to facilitate elution of albumin still present in the treated sample. Bilirubin species eluting from the column were detected by absorbance at 450 nm. Results of a number of chromatographic separations of pathological sera indicated a wide variation in the relative proportions of the four bilirubin fractions observed. A correlation of the sum of the areas of the bilirubin peaks observed by HPLC was found with the total bilirubin value obtained by a standard reference procedure.
已开发出一种高效反相液相色谱（HPLC）方法，用于分离胆汁中至少三种主要胆红素组分以及人血清中的四种组分。该方法与大多数其他方法不同，在于血清在注入HPLC柱之前并未完全脱蛋白；相反，血清用过量的硫酸钠溶液处理，主要沉淀大于白蛋白的蛋白质。将过滤并稀释后的上清液注入反相柱，然后在24分钟的梯度洗脱过程中实现胆红素种类的分离。初始水相酸性流动相和最终异丙醇基流动相均含有5%的甲氧基乙醇（v/v），以促进处理后样品中仍存在的白蛋白的洗脱。从柱中洗脱的胆红素种类通过在450nm处的吸光度进行检测。对多种病理血清的色谱分离结果表明，观察到的四种胆红素组分的相对比例存在很大差异。发现通过HPLC观察到的胆红素峰面积总和与通过标准参考程序获得的总胆红素值之间存在相关性。
