Liao Hsiao-Wei, Chen Guan-Yuan, Wu Ming-Shiang, Liao Wei-Chih, Tsai I-Lin, Kuo Ching-Hua
School of Pharmacy, College of Medicine, National Taiwan University, Taiwan.
School of Pharmacy, College of Medicine, National Taiwan University, Taiwan; The Metabolomics Core Laboratory, Center of Genomic Medicine, National Taiwan University, Taiwan.
J Chromatogr A. 2015 Jan 2;1375:62-8. doi: 10.1016/j.chroma.2014.11.073. Epub 2014 Dec 10.
Quantification of endogenous metabolites has enabled the discovery of biomarkers for diagnosis and provided for an understanding of disease etiology. The standard addition and stable isotope labeled-internal standard (SIL-IS) methods are currently the most widely used approaches to quantifying endogenous metabolites, but both have some limitations for clinical measurement. In this study, we developed a new approach for endogenous metabolite quantification by the postcolumn infused-internal standard (PCI-IS) method combined with the matrix normalization factor (MNF) method. MNF was used to correct the difference in MEs between standard solution and biofluids, and PCI-IS additionally tailored the correction of the MEs for individual samples. Androstenedione and testosterone were selected as test articles to verify this new approach to quantifying metabolites in plasma. The repeatability (n=4 runs) and intermediate precision (n=3 days) in terms of the peak area of androstenedione and testosterone at all tested concentrations were all less than 11% relative standard deviation (RSD). The accuracy test revealed that the recoveries were between 95.72% and 113.46%. The concentrations of androstenedione and testosterone in fifty plasma samples obtained from healthy volunteers were quantified by the PCI-IS combined with the MNF method, and the quantification results were compared with the results of the SIL-IS method. The Pearson correlation test showed that the correlation coefficient was 0.98 for both androstenedione and testosterone. We demonstrated that the PCI-IS combined with the MNF method is an effective and accurate method for quantifying endogenous metabolites.
内源性代谢物的定量分析有助于发现用于诊断的生物标志物，并增进对疾病病因的理解。标准加入法和稳定同位素标记内标（SIL-IS）法是目前用于定量分析内源性代谢物的最广泛使用的方法，但两者在临床测量中都存在一些局限性。在本研究中，我们开发了一种新的内源性代谢物定量方法，即柱后注入内标（PCI-IS）法与基质归一化因子（MNF）法相结合。MNF用于校正标准溶液与生物流体之间的基质效应（MEs）差异，而PCI-IS则进一步针对单个样品对MEs进行校正。选择雄烯二酮和睾酮作为测试对象，以验证这种定量血浆中代谢物的新方法。在所有测试浓度下，雄烯二酮和睾酮峰面积的重复性（n = 4次运行）和中间精密度（n = 3天）的相对标准偏差（RSD）均小于11%。准确度测试表明回收率在95.72%至113.46%之间。采用PCI-IS与MNF法相结合对50份来自健康志愿者的血浆样品中的雄烯二酮和睾酮浓度进行了定量，并将定量结果与SIL-IS法的结果进行了比较。Pearson相关性检验表明，雄烯二酮和睾酮的相关系数均为0.98。我们证明，PCI-IS与MNF法相结合是一种有效且准确的内源性代谢物定量方法。
