Optimization of GC-MS run time for untargeted metabolomics: Trade-offs between speed, coverage, and repeatability

Optimizing run time in gas chromatography-mass spectrometry (GC-MS) based metabolomics is essential for balancing metabolite coverage, reproducibility, and practical workflow constraints. In this study, three GC-MS methods with different run times, short (26.7 min), a standard method based on the established Fiehn protocol (37.5 min), and long (60 min), were evaluated across three biological matrices: cell culture, plasma, and urine. All methods were applied using identical injection volumes and derivatization protocols. The number of annotated metabolites in the short and standard methods was comparable: 138 vs. 156 in cell culture, 147 vs. 168 in plasma, and 186 vs. 198 in urine. The long method provided higher metabolite coverage (196 in cell culture, 175 in plasma, 244 in urine), largely due to improved chromatographic resolution and deconvolution, which also increased the number of unannotated features. Although the proportion of high-filling (0.75-1) annotated metabolites was similar across all methods (∼79-90 %), repeatability was slightly better in the standard and long methods (RSD ∼20-24 %) than in the short method (RSD ∼23-30 %). Notably, since derivatized samples must be analyzed within 24 h, the short method presents a practical advantage by enabling completion of full batch analysis within this time constraint. Overall, while the short and standard methods offer similar identification performance, the long method enhances analytical depth.