Quantitative analysis of trace-level bleomycin in complex matrices: Application to in vitro electrochemotherapy

Bleomycin, a cytotoxic antibiotic, poses substantial challenges for mass spectrometry-based analysis due to its extreme polarity, chelating properties, heterogeneity of fractions, and propensity to form multiple charged species during electrospray ionization. As one of the few effective drugs used in electrochemotherapy, the ability to quantify trace levels of bleomycin is critical for evaluating treatment efficacy, often requiring sensitivity beyond the capabilities of existing analytical methods. Such precise quantification would facilitate the evaluation of electrochemotherapy efficacy, such as comparing the in vitro effects of nanosecond electric pulses with conventional microsecond pulses. To address these challenges, we integrated cell viability assays with a robust chemical analytical approach. This approach employed solid-phase extraction for sample preparation, combined with HILIC-LC-MS/MS, achieving exceptional sensitivity with LLOQ of 0.075 µg/L and overcoming analyte and matrix complexity. Although a significant reduction in cell survival was confirmed when combining nanosecond pulses (25 × 400 ns) with bleomycin, chemical analysis revealed discrepancies, underscoring the complex interaction between electric pulse parameters and drug action. These findings highlight the need for further refinement of treatment protocols and the development of advanced analytical techniques.