Study of Metabolite Detectability in Simultaneous Profiling of Amine/Phenol and Hydroxyl Submetabolomes by Analyzing a Mixture of Two Separately Dansyl-Labeled Samples

Abstract

Background: Liquid chromatography-mass spectrometry (LC-MS), widely used in metabolomics, is often limited by low ionization efficiency and ion suppression, which reduce overall metabolite detectability and quantification accuracy. To address these challenges, chemical isotope labeling (CIL) LC-MS has emerged as a powerful approach, offering high sensitivity, accurate quantification, and broad metabolome coverage. This method enables comprehensive profiling by targeting multiple submetabolomes. Specifically, amine-/phenol- and hydroxyl-containing metabolites are labeled using dansyl chloride under distinct reaction conditions. While this strategy provides extensive coverage, the sequential analysis of each submetabolome reduces throughput. To overcome this limitation, we propose a two-channel mixing strategy to improve analytical efficiency.

Methods: In this approach, samples labeled separately for the amine/phenol and hydroxyl submetabolomes are combined prior to LC-MS analysis, leveraging the common use of dansyl chloride as the labeling reagent. This integration effectively doubles throughput by reducing LC-MS runtime and associated costs. The method was evaluated using human urine and serum samples, focusing on peak pair detectability and metabolite identification. A proof-of-concept study was also conducted to assess the approach's applicability in putative biomarker discovery.

Results: Results demonstrate that the two-channel mixing strategy enhances throughput while maintaining analytical robustness.

Conclusions: This method is particularly suitable for large-scale studies that require rapid sample processing, where high efficiency is essential.

Keywords: chemical isotope labeling (CIL) LC-MS; comprehensive metabolome profiling; dansyl labeling; rapid sample processing.

Figure 1

Workflow of the two-channel mixing-based CIL LC/MS analysis.

Figure 2

Venn diagrams illustrating the number of detected peak pairs in the A channel (red), H channel (blue), and AH mixture (purple) for urine (A–C) and serum (D–F) samples. In each channel, a labeled metabolite is detected as a co-eluting ¹²C/¹³C-labeled ion pair (i.e., peak pair) that exhibits the expected mass difference of 2.0067 Da. Overlapping and unique peak pairs were determined by comparing m/z and retention time across different channels. A peak pair was considered overlapping if both m/z and retention time matched within a defined tolerance window.

Figure 3

Percentage of peak pair loss in AH mixture across five intensity regions (0 to >500,000) for urine (A) and serum (B) samples. Peak pair loss is defined as peak pairs detected in individual A channels or H but absent in the AH mixture after sample mixing.

Figure 4

Distribution of peak pairs detected in the A channel, H channel, and AH mixture across five intensity ranges (from 0 to >500,000) for urine (A) and serum (B) samples. Each peak pair represents a labeled metabolite, detected as a co-eluting ¹²C/¹³C-labeled ion pair.

Figure 5

Mass spectra of a urine sample at 2.01 min for the A channel (A) and AH mixture (B). A region with peak pair loss is marked by a red * and enlarged for clarity: A channel enhanced (C) and AH mixture enhanced (D).

Figure 6

Mass spectra of a urine sample at 8.01 min for the H channel (A) and AH mixture (B). A region with peak pair loss is marked by a red * and enlarged for clarity: H channel enhanced (C) and AH mixture enhanced (D).

Figure 7

Venn diagram showing the number of identified metabolites in the A channel (red), H channel (blue), and AH mixture (purple) for urine (A,B) and serum (C,D) samples.

Figure 8

Volcano plots of binary comparisons of urine samples from two independent donors (Donor 1, Blue ; Donor 2, Red) for the A channel (A), H channel (B), and AH mixture (C). Venn diagram (D) illustrating the overlap of significantly changed metabolites (tier 1 and tier 2 only) among the A (red), H (blue), and AH (purple) channels.

Figure 9

Principal component analysis of urine samples from two independent donors for the A channel (A), H channel (B), and AH mixture (C).