Negative ion mode results. In alignment with the positive mode analysis, we performed similar comparisons using the negative ion mode data. To investigate cell metabolites changed due to the omission of LN2 quenching, we investigated the SCMS data obtained from cells in Group 1 vs. Group 2 as well as Groups 3 vs. Group 4. For comparison between Group 1 and Group 2, 145 metabolites were significantly changed with 20 increased and 125 decreased metabolites (Figure ). Pathway analysis revealed that galactose metabolism (Figure ) significantly changed (Table ). For comparison between Group 3 and Group 4, our results show that the abundances of 291 metabolites were significantly altered (Figure ), with 235 increased and 56 decreased metabolites. Using MS/MS analysis at the single-cell level, we identified multiple metabolites, including increased oleic acid and fatty acid FA (17:3) as well as deceased triglycerides TG (51:14) (Figure ) (Table ). Tentatively labeled species include decreased lipids (e.g., phosphatidylglycerols (PG(33:4), PG(43:4), PG(33:5), PG(32:4)), DG (38:5), DG (35:4), and sphingomyelins (SM(36:5), SM(36:6)) and increased organic acids (linoleic acid, succinic acid, and octadecenoic acid), lipid (MG (22:4)), and other small molecules (alpha-D-glucose and creatine). Significantly altered metabolites in the comparison of Groups 3 and Group 4 indicate substantially affected pathways, suggesting that storing samples at -80 °C without quenching is insufficient to preserve metabolomic integrity. Analysis of tentatively labeled metabolites revealed that 11 metabolic pathways were significantly affected due to storage without quenching (Table ). These pathways include alanine, aspartate, and glutamate metabolism, D-amino acid metabolism, butanoate metabolism, linoleic acid metabolism, galactose metabolism, arginine and proline metabolism, valine, leucine, and isoleucine biosynthesis, valine, leucine, and isoleucine degradation, glycine, serine, and threonine metabolism, pantothenate and CoA biosynthesis, and caffeine (Figure ).