High tolerance to instrument drifts by differential chemical isotope labeling LC‐MS: A case study of the effect of LC leak in long‐term sample runs on quantitative metabolome analysis

Abstract

Metabolomics study of a biological system often involves the analysis of many comparative samples over a period of several days or weeks. This process of long-term sample runs can encounter unexpected instrument drifts such as small leaks in liquid chromatography-mass spectrometry (LC-MS), degradation of column performance, and MS signal intensity change. A robust analytical method should ideally tolerate these instrumental drifts as much as possible. In this work, we report a case study to demonstrate the high tolerance of differential chemical isotope labeling (CIL) LC-MS method for quantitative metabolome analysis. In a study of using a rat model to examine the metabolome changes during rheumatoid arthritis (RA) disease development and treatment, over 468 samples were analyzed over a period of 15 days in three batches. During the sample runs, a small leak in LC was discovered after a batch of analyses was completed. Reanalysis of these samples was not an option as sample amounts were limited. To overcome the problem caused by the small leak, we applied a method of retention time correction to the LC-MS data to align peak pairs from different runs with different degrees of leak, followed by peak ratio calculation and analysis. Herein, we illustrate that using 12 C-/ 13 C-peak pair intensity values in CIL LC-MS as a measurement of concentration changes in different samples could tolerate the signal drifts, while using the absolute intensity values (ie, 12 C-peak as in conventional LC-MS) was not as reliable. We hope that the case study illustrated and the method of overcoming the small-leak-caused signal drifts can be helpful to others who may encounter this kind of situation in long-term sample runs.

Evaluating and minimizing batch effects in metabolomics

“…The QC sample was prepared by mixing an equal mole amount of the 12 C‐labeled and 13 C‐labeled pooled samples and injected into liquid chromatography‐mass spectrometry after every 10 sample runs. A total of 468 rat plasma samples were analyzed over a period of 15 days in three batches (Adapted with permission from Chen et al 2020b) [Color figure can be viewed at wileyonlinelibrary.com]…”

“…After the individual sample is mixed with the heavy‐isotope‐labeled reference sample, the pair of labeled metabolites will experience the same analytical variations, and thus the relative quantification will not be affected anymore. Figure 5 shows an example of a differentially chemical‐isotope‐labeled metabolite detected in two batches (some of the runs in batch 1 had encountered a small leak in LC) (Chen et al, 2020b). The absolute intensities of peaks shown in the mass spectra were changed, whereas the peak ratio remained to be similar.…”