ThermoPlate system. A single cell was selected by the cell selection probe, and the cell diameter was measured using the inverted microscope. In fact, the microscope enables the discrimination between cancerous and non-cancerous cells based on their morphological characteristics. The cell was then transferred to the Single-probe tip, where the cell was immediately lysed when contacting the solvent (e.g., acetonitrile containing the internal standard). The single cell lysate and the internal standard were simultaneously detected by MS. Bensen et. al accurately measured intracellular amounts and concentrations of the chemotherapy drug gemcitabine in individual bladder cancer cells, including both K562 cell lines and bladder cancer cells isolated from patients undergoing chemotherapy . Comparisons with traditional LC/MS results of K562 cells yielded comparable intracellular drug concentrations. This study demonstrates the system's capacity for real-time, precise quantification of anticancer drug levels in single cells, highlighting its potential for improving personalized chemotherapy regimens. A key advantage of these dicationic compounds lies in their selectivity for complex formation, allowing the discrimination of low-abundance ions with nearly identical m/z values. Additionally, MS/MS was employed for molecular identification of selected adduct ions. This reactive SCMS method represents a significant advancement by enabling the simultaneous detection of negatively and positively charged metabolites in a single experiment. Most notably, many of the negatively charged metabolites identified using dicationic reagents were undetectable in negative ionization mode alone, demonstrating the enhanced sensitivity offered by this technique. Future studies could explore other compounds to further refine the sensitivity and scope of metabolite detection in single-cell analysis. . NO, a reactive and short-lived molecule (with a half-life of less than one second), plays a critical role in various biological processes, including angiogenesis in tumors. Quantifying NO at the single-cell level remains challenging due to the small size of cells and NO's reactive nature. There are two main pathways for NO production: exogenous (provided by NO donor compounds) and endogenous (produced by cells). Clinically, NO donors are used in the treatment of conditions like high blood pressure and heart disease. Additionally, the anticancer drug doxorubicin (DOX) can increase endogenous NO levels via the catalytic activity of nitric oxide synthase (NOSs). Given NO's crucial biological functions, developing a method to accurately quantify NO at the single-cell level is highly significant. Lan et al. proposed a method based on a quantitative reaction between NO and amlodipine (AML), a compound containing the Hantzsch ester group. The reaction between NO and AML yields dehydroamlodipine (DAM), which can then be detected and quantified using the Single-probe SCMS technique. Importantly, AML reacts selectively with NO, exhibiting 100% efficiency without interference from other reactive species within the cell. In their study, individual cells were adhered to glass chips containing microwells and were subsequently treated with AML under different experimental conditions. To induce NO production, two compounds were used: sodium nitroprusside (SNP) (to generate exogenous NO) and doxorubicin (DOX) (to stimulate production of endogenous NO). The Single-probe SCMS system was employed for NO quantification, with acetonitrile (ACN) containing 0.1% formic acid (FA) and 1.0 μM OXF (as an internal standard) used as the sampling solution. Results from the SCMS studies demonstrated that intracellular NO levels exhibited heterogeneous distributions across the treated cells under all experimental conditions. This method provides a robust approach for the quantification of NO at the single-cell level, offering insights into the complex biological roles of NO in cellular systems.