One-Sample Calibration Curve on GC-MS Using Silicon Isotopologue Channels from Silylation Derivatization: Validation with Nonvolatile Organic Acids

Abstract Quantitative GC-MS analysis of nonvolatile metabolites conventionally requires multisample external calibration, imposing substantial preparative overhead in high-throughput settings. Here, the integration of silylation derivatization with an isotopologue-based one-sample calibration curve (OSCC) on GC-MS platforms is reported. Trimethylsilylation introduces silicon atoms with invariant natural isotope abundances (28Si:29Si:30Si = 92.23%:4.68%:3.09%), enabling multiple calibration anchors from a single standard injection. Two strategies were developed: OSCC with multiple isotopologue selected ion monitoring (OSCC-MISM) on GC-MS, serving as a compositional-verification precursor, and OSCC with multiple isotopologue reaction monitoring (OSCC-MIRM) on GC-MS/MS, recommended for real-sample quantification. Both were validated against multisample calibration curve (MSCC)-SIM and MSCC-MRM across 12 nonvolatile organic acids, achieving R2 > 0.99, RSDs below 16.1%, and recoveries of 80.5–111.2%. In 15 commercial Baijiu samples, Bland–Altman analysis of 180 paired measurements gave a pooled bias of +0.8%, with 95% limits of agreement within the prespecified ±15% margin, supporting equivalence of OSCC-MIRM with MSCC-MRM.