Retention time shift analysis and correction in chemical isotope labeling liquid chromatography/mass spectrometry for metabolome analysis.

Abstract

In chemical isotope labeling (CIL) liquid chromatography/mass spectrometry (LC/MS) metabolome analysis, the peak pairs of the same metabolite detected from different samples are aligned according to their mass and retention time (RT). Any RT shift of a peak pair in one of the sample files that falls outside the tolerance window will result in misalignment of the pair as a different metabolite. Thus, determination and correction of any significant RT shift are important to ensure the generation of high-quality metabolome results. In CIL LC/MS, the heavy-isotope-labeled pooled sample is spiked into all light-isotope-labeled individual samples. As a result, in the analysis of labeled samples of the same type, many common metabolites are detectable with high intensity in all LC/MS runs. We have developed a method to select a few of these metabolites as internal RT reference markers to check the occurrence of any RT shift in an LC/MS run. If a significant shift is found, an expanded list of these markers with their RT values covering the entire LC RT window is selected to serve as internal RT calibrants to recalibrate the chromatogram to correct any RT shift. We developed a software program in R to perform RT check (RTC) and recalibration (RT-calib). This program can quickly determine the occurrence of any RT shift falling outside a user-defined threshold in an LC/MS run, thereby triggering a timely intervention to correct the problem (e.g., fixing a small leak or changing a column). In the analysis of 278 dansylation LC/MS runs of human urine samples, we show that the RT values can be corrected to be within a 30-second window. An RT-check method and program tailored to CIL LC/MS metabolome analysis have been developed for quick detection and correction of RT shifts during the course of running many metabolome samples.