Lc ms eic plot interpretation

LC-MS EIC Plot Interpretation

Summary

Visual inspection and validation of extracted ion chromatogram (EIC) plots to confirm successful target detection, peak localization within expected m/z and retention time windows, and integration quality in targeted LC-MS metabolomics workflows. This skill bridges automated peak detection (via TARDIS) and quality assessment by examining diagnostic EIC plots for visibility, correct elution timing, and peak morphology.

When to use

After executing TARDIS in screening_mode = TRUE on centroided .mzML files with a defined target compound list. Use this skill when you need to verify that all target compounds are visually detectable within their specified m/z and retention time windows before committing to full-run peak detection and quantification. Critical for validating window parameters and identifying compounds that elute at retention time boundaries (e.g., early or late elution requiring window adjustment).

When NOT to use

• Input is already a feature table or quantification results table — interpretation applies to raw EIC plots, not aggregated data.
• Screening mode has not been executed or EIC plot files are not available — this skill requires diagnostic visual outputs from TARDIS.
• Data are in profile (not centroided) mode or in non-mzML formats — TARDIS screening_mode requires centroided .mzML input.

Inputs

• TARDIS screening_mode output folder containing diagnostic EIC plots (images or .pdf files)
• Target compound metadata data.frame with columns: compound ID, Name, theoretical m/z, expected retention time (minutes), ionization polarity
• Centroided .mzML LC-MS data files (already processed by TARDIS Spectra object loading)

Outputs

• Visual assessment report documenting presence/absence and localization of each target EIC within m/z and RT windows
• List of targets requiring m/z or retention time window adjustment before full quantification
• Confirmation that all 10 targets are visible and correctly localized (or identification of problematic targets)

How to apply

Load and examine the diagnostic EIC plots saved by TARDIS in the screening output folder. For each of the 10 (or N) target compounds, visually confirm: (1) the target signal is present and distinguishable from baseline noise within the defined m/z tolerance window; (2) the peak elutes within the expected retention time window, noting compounds that elute toward window edges (e.g., targets 1577 and 1583); (3) peak morphology is consistent with a single, well-integrated peak (not split or heavily distorted by instrument scan patterns). Document any targets with missing or mislocalized signals as requiring window parameter re-optimization. This visual triage prevents wasteful full-run quantification on poorly parameterized targets and informs retention time correction calibration.

Related tools

• TARDIS (Generates diagnostic EIC plots in screening_mode and performs automated polarity filtering and empty spectra removal; user interprets output plots to validate target detection and window parameters) — https://github.com/pablovgd/TARDIS
• Spectra (Loads centroided .mzML MS data as objects for TARDIS processing; enables polarity and spectrum metadata filtering upstream of EIC plot generation)
• xcms (Provides retention time correction algorithm integrated into TARDIS; supports validation of expected retention times used in EIC window definitions)

Examples

# After TARDIS screening_mode = TRUE execution:
# Visually inspect EIC plots in output folder; verify all targets present within m/z ± tolerance and expected RT; document targets with edge elution (e.g., 1577, 1583) for window refinement.

Evaluation signals

• All 10 target compounds are visually present in their corresponding EIC plots with signal-to-baseline ratio sufficient to distinguish peak from noise.
• Each target peak is localized within ±tolerance of its expected m/z and retention time window; compounds eluting at window edges are flagged for parameter review.
• Peak morphology is single, well-defined (not split or sawtooth-distorted); sawtooth patterns indicate empty spectra were not fully filtered by TARDIS.
• No targets are missing or completely absent from plots — if absent, confirm target is in the input data.frame and ionization polarity is correct.
• Retention time localization is reproducible across replicate runs or QC samples (if available) — large shifts between runs suggest RT correction calibration is needed.

Limitations

• Visual interpretation is subjective and may vary by analyst; no quantitative acceptance criteria are specified in the article. Peak visibility depends on compound ionization efficiency and instrument sensitivity, not only window parameters.
• Sawtooth peak profiles may occur in data with multiple overlapping m/z scan windows even after TARDIS empty spectra filtering, complicating visual assessment of peak integration quality.
• EIC plot resolution and dynamic range settings (not detailed in the article) affect visibility of low-abundance targets; plots must be examined at consistent zoom/contrast to enable fair comparison.
• Screening mode validates target detection in a subset of runs; full quantification may still fail if target signals drop below detection limit in some samples or QC runs are not included.

Evidence

• [other] The screening mode run with tardisPeaks produced diagnostic EIC plots showing successful detection and integration for all 10 targets: "The screening mode run with tardisPeaks produced diagnostic EIC plots showing successful detection and integration for all 10 targets, with visual confirmation that targets 1577 and 1583 elute toward"
• [intro] First, we perform a screening step to check if our targets are visible within our m/z and RT windows: "First, we perform a screening step to check if our targets are visible within our m/z and RT windows"
• [other] Inspect the resulting EIC plots saved in the screening output folder to verify each target is visible and correctly localized within its m/z and retention-time window: "Inspect the resulting EIC plots saved in the screening output folder to verify each target is visible and correctly localized within its m/z and retention-time window."
• [results] The resulting EICs are again saved in the output folder and can be inspected: "The resulting EICs are again saved in the output folder and can be inspected"
• [intro] you will notice that peaks will have a sawtooth profile, because of the filtering of empty spectra within TARDIS: "you will notice that peaks will have a sawtooth profile, because of the filtering of empty spectra within TARDIS"