Department of Lipidomics, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113-8654, Japan.
Department of Clinical Laboratory Medicine, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113-8654, Japan.
Rapid Commun Mass Spectrom. 2020 Jul 15;34(13):e8814. doi: 10.1002/rcm.8814.
The electrospray ionization mass spectrometry (ESI-MS) methodology often shows poor ionization reproducibility in the analysis of biological samples. Therefore, normalization of the measured peak intensities is essential. It is believed that quantitative data with high reproducibility can be obtained by adding a constant amount of an internal standard (IS) material labeled with stable isotopes to each sample, thus allowing the correction of the quantitative value of the target compound by that of the IS. We investigated whether the presence or absence of a labeled IS improves the accuracy of these quantitative values.
Triple quadrupole MS coupled with liquid chromatography was used to analyze fatty acid metabolites in biological samples as target compounds. Two independent systems were used to provide a measure of reproducibility in two different laboratories.
Data having poor reproducibility in the raw peak areas were efficiently normalized using the IS, but, crucially, the IS method using stable isotopes was not always necessary. In some cases, the reproducibility was relatively good even without using the IS. In a contaminant matrix, the MS response behavior of the target compound and its stable isotope-labeled material was complicated. Since ion suppression by matrix contaminants was dependent on the concentration of the target compound, the added amounts of the ISs were also important, Furthermore, an equivalent normalization effect was obtained by using a pooled quality control sample as an external standard, thus obviating the need for labeled IS samples, which are often expensive and sometimes not commercially available.
Our results raise the question as to whether the quantitative method using stable-isotope-labeled ISs is always necessary and beneficial. However, the results obtained in this study cannot be generalized because only fatty acid metabolites were examined using ESI-MS and only a highly substituted deuterium-labeled IS was used.
电喷雾电离质谱（ESI-MS）方法在分析生物样品时经常显示出较差的离子化重现性。因此，必须对测量的峰强度进行归一化。人们认为，通过向每个样品中添加一定量用稳定同位素标记的内标（IS）物质，可以获得重现性高的定量数据，从而可以通过 IS 的定量值来校正目标化合物的定量值。我们研究了是否存在标记的 IS 会提高这些定量值的准确性。
采用串联三重四极杆质谱仪与液相色谱联用的方法，分析生物样品中的脂肪酸代谢产物作为目标化合物。使用两个独立的系统在两个不同的实验室提供重现性的衡量标准。
使用 IS 可以有效地归一化原始峰面积重现性差的数据，但重要的是，并非总是需要使用稳定同位素的 IS 方法。在某些情况下，即使不使用 IS，重现性也相对较好。在污染物基质中，目标化合物及其稳定同位素标记材料的 MS 响应行为很复杂。由于基质污染物的离子抑制作用取决于目标化合物的浓度，因此添加的 IS 量也很重要。此外，使用混合质量控制样品作为外部标准也可以获得等效的归一化效果，从而无需使用通常昂贵且有时无法从商业途径获得的标记 IS 样品。
我们的结果提出了一个问题，即使用稳定同位素标记 IS 的定量方法是否总是必要和有益。然而，由于仅使用 ESI-MS 检查了脂肪酸代谢产物，并且仅使用高度取代的氘标记 IS，因此本研究的结果不能推广。
