Translating 2D-Chromatographic Fingerprinting to Quantitative Volatilomics: Unrevealing Compositional Changes in Maize Silage Volatilome for Robust Marker Discovery

This study examines the complex volatilome of maize silage, both with and without commercial heterolactic strain inoculation, conserved for 100 days, using quantitative volatilomics. Chemical classes linked to microbial metabolism were analyzed across a concentration range from 10 μg g^–1 to 1 ng g^–1. A reference method using comprehensive two-dimensional gas chromatography (GC × GC) and time-of-flight mass spectrometry (TOF MS) with loop-type thermal modulation (TM) was translated to a differential-flow modulation (FM) platform with parallel MS and flame ionization detector (FID) detection. With translation, the original method’s analyte elution order and resolution are preserved. The new method allowed for accurate quantification using multiple headspace solid-phase microextraction (MHS-SPME) and FID-predicted relative response factors (RRFs). Both methods showed comparable discriminatory power with FM GC × GC-MS/FID achieving satisfactory quantification accuracy without external calibration. Analysis of 98 volatiles provided insights into silage fermentation, supporting marker discovery and correlations with silage quality and stability.