In this study, live HCT-116 cells were washed by ammonium formate solution, quenched by LN2, freeze-dried in a vacuum, and stored in a -80 ℃ freezer. We then performed single-cell metabolomics studies using the Single-probe SCMS technique. Our results indicated that washing using ammonium formate led to enhancement in ion intensities attributed to the mitigated matrix effect. Remarkably, a diverse array of lipids, including PC, PS, PE, PA, PG, TG, DG, and MG were identified from individual cells. We further studied the influence of LN2 quenching and storage at -80 °C on metabolites and metabolomic pathways. Notably, LN2 quenching and freeze-drying preserved cells' metabolomic profiles. Storage of LN2 quenched cells at -80 ℃ for 48 h generally retained cell metabolites, enabling reliable SCMS experiments with extended time or low temperature shipped samples. However, the time delay between LN2 quenching and SCMS experiments should be minimized. Although cells underwent rapid drying in vacuum at room temperature could largely retain metabolites, cells need to be immediately analyzed because storage (even at -80 ℃ for 48 h) could change metabolites' compositions. These findings collectively contribute to the sample preparation techniques in single-cell metabolomics studies. The developed methods can be readily adopted by researchers using other ambient-based SCMS techniques for broad applications.