de Korte D, Haverkort W A, Roos D, van Gennip A H
Clin Chim Acta. 1985 Jun 14;148(3):185-96. doi: 10.1016/0009-8981(85)90145-7.
An anion-exchange high performance liquid chromatography (HPLC) method is described for the quantitation of intracellular purine and pyrimidine nucleotides. With an ammonium phosphate salt and pH gradient, complete separation is achieved of all major nucleotides and several interfering substances, such as dehydroascorbic acid and NAD. For optimal resolution of the monophosphates, strict control of the equilibration pH is essential. To prevent interference by a degradation product of NADPH with the determination of GDP, the pH of the high-ionic strength buffer has to be in the range of 4.9-5.0. The use of radially compressed, prepacked cartridges filled with Partisil-10 SAX appeared to be a fast and cheap alternative for expensive stainless-steel columns. The use of ammonium phosphate buffers, in combination with precolumns filled with pellicular silica and SAX resin, and interim EDTA washes prevents baseline shift. This allows analysis at 0.01 Absorbance Units Full Scale during the entire column lifetime (about 180 analyses), which is sufficiently sensitive for the quantitation of low levels of nucleotides, especially when the amount of sample is limited. The usefulness of the presented chromatographic system is demonstrated by the quantitation of the nucleotides in extracts of lymphocytes and neutrophils from the blood of healthy human donors. With this method nucleotide concentrations were measured, with a within-assay variation of 5-10% and an inter-donor variation of 10%.
本文描述了一种用于定量细胞内嘌呤和嘧啶核苷酸的阴离子交换高效液相色谱（HPLC）方法。通过磷酸铵盐和pH梯度，可实现所有主要核苷酸与几种干扰物质（如脱氢抗坏血酸和NAD）的完全分离。为了实现单磷酸盐的最佳分离效果，严格控制平衡pH至关重要。为防止NADPH的降解产物干扰GDP的测定，高离子强度缓冲液的pH必须在4.9 - 5.0范围内。使用填充有Partisil - 10 SAX的径向压缩预装柱，对于昂贵的不锈钢柱来说，似乎是一种快速且廉价的替代方案。使用磷酸铵缓冲液，结合填充有薄壳硅胶和SAX树脂的预柱以及中间的EDTA冲洗，可防止基线漂移。这使得在整个柱寿命期间（约180次分析）能够以0.01吸光度单位满量程进行分析，对于低水平核苷酸的定量分析而言，该灵敏度足够高，特别是当样品量有限时。通过对健康人类供体血液中淋巴细胞和中性粒细胞提取物中的核苷酸进行定量分析，证明了所提出的色谱系统的实用性。采用该方法测量核苷酸浓度，批内变异为5 - 10%，供体间变异为10%。
