Journal
ANALYTICAL BIOCHEMISTRY
Volume 683, Issue -, Pages -Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2023.115333
Keywords
IITZ-01; Lysosomotropic autophagy inhibitor; LC-MS/MS; Bioanalytical method development and; validation
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This study developed a bioanalytical method for quantification of IITZ-01 and evaluated its pharmacokinetics and metabolic stability in mice. The method showed good accuracy and precision, making it suitable for pre-clinical investigation.
The present study evaluates the pharmacokinetics and metabolic stability of a novel lysosomotropic autophagy inhibitor, IITZ-01 using an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS). It is required as this lead molecule awaits pre-clinical studies for development because of significant therapeutic outcomes in triple-negative breast cancer and renal cancer. A bioanalytical method for the quantitative determination of IITZ-01 in the plasma of mice was developed using the UPLC-MS/MS technique. The UPLC-MS/MS method was validated according to US-FDA bioanalytical guidance and successfully applied to study the pharmacokinetics and metabolic stability. Separation of IITZ- 01 and ZSTK474 (IS) from endogenous components with high selectivity and sensitivity (0.5 ng/mL) was achieved using Waters Acquity BEH C-18 column (50 mm x 2.1 mm, 1.7 mu m). A gradient mobile phase consisting of 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile was applied at a flow rate of 0.2 mL/min. Electrospray ionization was employed in positive ion mode for detection, while quantification utilized the multiple reaction monitoring (MRM) mode. This involved using [M+H]+fragment ions at m/z 483.19 -* 235.09 for IITZ-01 and m/z 418 -* 138 for the internal standard (IS). The method was validated over the calibration range of 0.5-800 ng/mL. The LLOQ of IITZ01 was 0.5 ng/mL in mice plasma. The method demonstrated good in terms of intra- and inter-day precision and accuracy. The matrix effect was found to be negligible, and the stability data were within acceptable limits. The validated technique supports suitability, reliability, reproducibility, and sensitivity for the pre-clinical investigation of IITZ-01 pharmacokinetics in mice and metabolic stability in human liver microsomes.
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