Holden Marcia J, Rabb Savelas A, Tewari Yadu B, Winchester Michael R
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, Maryland 20899-8311, USA. marcia.holden@ nist.gov
Anal Chem. 2007 Feb 15;79(4):1536-41. doi: 10.1021/ac061463b.
Measurement of the phosphorus content of nucleotides and deoxyribonucleic acid (DNA) offers an approach to the quantitation of nucleic acids that is traceable to the SI. Such measurements can be an alternative to the commonly used spectroscopic tools that are not traceable. Phosphorus measurements of thymidine 5'-monophosphate (TMP) and acid-digested plasmid and genomic DNA preparations were made using high-performance inductively coupled plasma optical emission spectroscopy (HP-ICP-OES) and high-performance liquid chromatography (HPLC) and compared for bias and uncertainty. A prerequisite for quality measurement is the purity of the materials. Quantitation with the two platforms was comparable for the TMP. However, the HPLC values had larger uncertainties and were all statistically different from the gravimetric values at the 95% confidence level. When using ICP-OES, the digestion of the nucleotide monophosphate can be eliminated, thus simplifying the procedure. The differences between the results obtained by using the two platforms, when measuring genomic or plasmid DNA, were dependent on the mass fraction of the digest. ICP-OES measurement of phosphorus provides a highly accurate quantitation for both nucleotide monophosphates and DNA with expanded uncertainties of less than 0.1%. Currently, ICP-OES requires a significant sample size restricting its usefulness for the quantitation of DNA but represents a valuable tool for certification of reference materials. HPLC requires smaller amounts of material to perform the analysis but is less useful for certification of reference materials because of lower accuracy and 10-fold higher expanded uncertainties.
核苷酸和脱氧核糖核酸（DNA）中磷含量的测量提供了一种可溯源至国际单位制（SI）的核酸定量方法。此类测量可替代常用的不可溯源的光谱工具。使用高效电感耦合等离子体发射光谱法（HP-ICP-OES）和高效液相色谱法（HPLC）对胸苷5'-单磷酸（TMP）以及酸消化的质粒和基因组DNA制剂进行磷含量测量，并比较其偏差和不确定度。质量测量的一个前提是材料的纯度。两种平台对TMP的定量结果具有可比性。然而，HPLC值的不确定度更大，并且在95%置信水平下，所有结果与重量法测量值在统计学上均有差异。使用ICP-OES时，可省去单磷酸核苷酸的消化步骤，从而简化了操作过程。测量基因组或质粒DNA时，两种平台所得结果的差异取决于消化物的质量分数。ICP-OES对磷的测量为单磷酸核苷酸和DNA都提供了高精度定量，扩展不确定度小于0.1%。目前，ICP-OES需要大量样品，这限制了其在DNA定量方面的应用，但它是参考物质认证的宝贵工具。HPLC进行分析所需的材料量较少，但由于准确度较低且扩展不确定度高10倍，在参考物质认证方面用处较小。
