LC–MS/MS Assay for Simultaneous Quantification of Dual‐Targeting 1,5‐Diaryl‐1,2,4‐Triazole Sulfonamides (WA11–13) in Human Plasma

Abstract

We recently designed and synthesized novel dual-targeting anticancer 1,5-diaryl-1,2,4-triazole-tethered sulfonamides. Among them, WA11-13 showed promising carbonic anhydrase and VEGFR-2 inhibitory activity. This study presents a validated, sensitive LC-MS/MS assay for quantifying these compounds in human plasma. Such assay would be very important for future preclinical studies and therapeutic drug monitoring. After protein precipitation with acetonitrile, analytes and the internal standard (carbamazepine) were separated on a Phenomenex Kinetex C18 column using binary gradient elution. The mobile phase was 0.1% formic acid in water and acetonitrile (95:5, v/v) at 0.7 mL/min. Total run time was under 6 min. Detection used an API 3500 triple quadrupole mass spectrometer with electrospray ionization in positive mode. Quantification relied on multiple reaction monitoring for high sensitivity and specificity. The method was fully validated per FDA guidelines, showing acceptable linearity, accuracy, precision, selectivity, and stability. Linearity was observed in the ranges of 2.5-750 ng/mL for WA11, 25-1000 ng/mL for WA12, and 50-1000 ng/mL for WA13. The method was successfully applied to spiked human plasma, supporting its potential for therapeutic drug monitoring. Validation confirms the assay's high sensitivity, accuracy, and precision, making it suitable for future preclinical and clinical investigations of these investigational anticancer agents.