Translating 2D-Chromatographic\nFingerprinting to Quantitative\nVolatilomics: Unrevealing Compositional Changes in Maize Silage Volatilome\nfor Robust Marker Discovery

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