Tsakali E, Petrotos K, Chatzilazarou A, Stamatopoulos K, D'Alessandro A G, Goulas P, Massouras T, Van Impe J F M
Department of Scienze Agro-Ambientali e Territoriali, Università degli Studi di Bari Aldo Moro, Via G. Amendola, 165/A, 70126 Bari, Italy.
Department of Biosystems Engineering, Technological Educational Institute of Larissa, Larissa 41110, Greece.
J Dairy Sci. 2014;97(8):4832-7. doi: 10.3168/jds.2013-7526. Epub 2014 May 23.
In the current paper, a method is introduced to determine lactoferrin in sweet whey using reversed-phase HPLC without any pretreatment of the samples or use of a separation technique. As a starting point, the most common HPLC protocols for acid whey, which included pretreatment of the whey along with a sodium dodecyl sulfate-PAGE step, were tested. By skipping the pretreatment and the separation steps while altering the gradient profile, different chromatographs were obtained that proved to be equally efficient to determine lactoferrin. For this novel 1-step reversed-phase HPLC method, repeatability was very high over a wide range of concentrations (1.88% intraday to 5.89% interday). The limit of detection was 35.46μg/mL [signal:noise ratio (S/N)=3], whereas the limit of quantification was 50.86μg/mL (S/N=10). Omitting the pretreatment step caused a degradation of the column's lifetime (to approximately 2,000 samples). As a result, the lactoferrin elution time changed, but neither the accuracy nor the separation ability of the method was significantly influenced. We observed that this degradation could be easily avoided or detained by centrifuging the samples to remove fat or by extensive cleaning of the column after every 5 samples.
在当前论文中，介绍了一种无需对样品进行任何预处理或使用分离技术即可通过反相高效液相色谱法测定甜乳清中乳铁蛋白的方法。作为起点，测试了用于酸乳清的最常见高效液相色谱方案，其中包括对乳清进行预处理以及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳步骤。通过跳过预处理和分离步骤，同时改变梯度曲线，获得了不同的色谱图，结果证明这些色谱图在测定乳铁蛋白方面同样有效。对于这种新颖的一步反相高效液相色谱法，在很宽的浓度范围内重复性都非常高（日内为1.88%，日间为5.89%）。检测限为35.46μg/mL [信噪比（S/N）=3]，而定量限为50.86μg/mL（S/N=10）。省略预处理步骤会导致色谱柱寿命缩短（至约2000个样品）。结果，乳铁蛋白的洗脱时间发生了变化，但该方法的准确性和分离能力均未受到显著影响。我们观察到，通过离心样品以去除脂肪或每5个样品后对色谱柱进行彻底清洗，可以轻松避免或延缓这种降解。
