Development of a quantitative LC-MS/MS-based assay for Lycosin-II-analog 3 and assessment of its stability in biological matrices.

Lycosin-II (L-II), a 21-amino acid peptide from Lycosa singoriensis venom, exerts antimicrobial activity via membrane depolarization and permeabilization. Subsequent studies identified the analog L-II-3 as a promising antimicrobial therapeutic. Given the pharmacokinetic limitations, such as metabolic instability, often encountered with peptide-based therapeutics, quantitative characterization of the biological stability and degradation mechanisms of L-II-3 is essential for its in vivo translation. Thus, this study aimed to develop and validate a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for L-II-3 in biological samples and to investigate its degradation mechanisms. Calibration standards in mouse plasma and whole blood were prepared by direct protein precipitation. The assay achieved acceptable accuracy and precision (<15%) across concentration ranges of 100-1000 nM in both mouse plasma and whole blood. The stability test indicated that L-II-3 was relatively stable in plasma (88.6 ± 6.2% remaining after 60 min) but rapidly disappeared in whole blood (half-life of 19.7 min). Among the various protease inhibitors tested, EDTA and phenanthroline partially prolonged the half-life of L-II-3 in whole blood. Further assessment using cytosolic and ghost membrane fractions prepared from red blood cells (RBCs) indicated that metal-ion-dependent enzymes in the cytosolic fraction are the primary contributors to L-II-3 degradation, with an additional contribution from RBC membrane binding. L-II-3 degradation was also enhanced by microbial proteases secreted from selected microbial strains. This validated LC-MS/MS assay provides a critical tool for upcoming in vivo pharmacokinetic studies of L-II-3 and its analogs, guiding strategies to enhance in vivo circulation time and therapeutic efficacy.