Wang Hsiaoling, Pampati Nagarani, McCormick William M, Bhattacharyya Lokesh
Division of Biological Standard and Quality Control, Office of Compliance and Biologics Quality, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993.
Division of Biological Standard and Quality Control, Office of Compliance and Biologics Quality, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993.
J Pharm Sci. 2016 Jun;105(6):1851-1857. doi: 10.1016/j.xphs.2016.03.039.
We report development and validation of a simple, rapid, and accurate method for the quantitation of protein nitrogen, which combines Kjeldahl digestion and ion chromatography with suppressed conductivity detection and requires nanomolar amount of nitrogen in samples (≥10 μg protein). The mechanism of suppressed conductivity detection does not permit analysis of samples containing copper (present in Kjeldahl digestion solution) and aluminum (present in many vaccines as adjuvants) due to precipitation of their hydroxides within the suppressor. We overcame this problem by including 10 μM oxalic acid in Kjeldahl digests and in the eluent (30 mM methanesulfonic acid). The chromatography is performed using an IonPac CS-16 cation exchange column by isocratic elution. The method reduces the digestion time to less than 1 h and eliminates the distillation and titration steps of the Kjeldahl method, thereby reducing the analysis time significantly and improving precision and accuracy. To determine protein nitrogen in samples containing non-protein nitrogen, proteins are precipitated by a mixture of deoxycholate and trichloroacetic acid and the precipitates are analyzed after dissolving in KOH. The method is particularly useful for biological samples that are limited and can also be applied to food, environmental, and other materials.
我们报告了一种用于蛋白质氮定量的简单、快速且准确的方法的开发与验证，该方法将凯氏定氮法消化与离子色谱结合，并采用抑制电导检测，样品所需氮量为纳摩尔级（≥10μg蛋白质）。抑制电导检测机制不允许分析含有铜（存在于凯氏消化液中）和铝（作为佐剂存在于许多疫苗中）的样品，因为它们的氢氧化物会在抑制器中沉淀。我们通过在凯氏消化液和洗脱液（30mM甲磺酸）中加入10μM草酸克服了这个问题。使用IonPac CS - 16阳离子交换柱通过等度洗脱进行色谱分析。该方法将消化时间缩短至不到1小时，消除了凯氏定氮法的蒸馏和滴定步骤，从而显著缩短了分析时间，提高了精密度和准确度。为了测定含有非蛋白质氮的样品中的蛋白质氮，用脱氧胆酸盐和三氯乙酸的混合物沉淀蛋白质，沉淀溶解在氢氧化钾中后进行分析。该方法对有限的生物样品特别有用，也可应用于食品、环境和其他材料。
