Development and Standardization of an <em>Ex Vivo</em> Micromethod for Intracellular Quantification of Vincristine in Primary ALL Cells by LC-MS/MS

Quantifying intracellular vincristine (VCR) in primary pediatric acute lymphoblastic leukemia (ALL) cells is essential for understanding sample-specific differences in drug uptake and for supporting experimental studies using scarce patient material. Here, we present a streamlined LC-MS/MS micromethod specifically optimized for primary ALL cells obtained through patient-derived xenografts (PDX), addressing key challenges such as limited cell availability, small cell size, and the need for reproducible recovery after multiple washing steps. Time-course assays ranging from 1 to 5 h demonstrated that a 3 h incubation period yields the highest and most consistent intracellular VCR levels, with lower variability across replicates. Method optimization also established that using 2.5 × 10⁶ cells per condition improves analytical robustness compared with 1 × 10⁶ cells, which showed greater dispersion in repeated experiments. Additional critical refinements included the use of cold methanol to enhance protein precipitation efficiency and the incorporation of an internal standard to monitor and minimize procedural variation. The method enables sensitive and precise quantification of intracellular VCR across relevant concentration ranges, although the lowest tested dose (0.01 µM) was not quantifiable after the 3 h incubation. Overall, this micromethod provides a practical, reproducible, and scalable approach for measuring intracellular VCR in primary ALL samples. Its design supports comparative analyses, validation of ex vivo models, and future methodological applications aimed at integrating intracellular drug quantification into broader pharmacological assessments.