Drug Plasma Stability of PG-P and Carbonic Anhydrase hybrids inhibitors by LC-MS/MS and application of LEDA algorithm for their characterization

Recently it has been shown that P-glycoprotein (P-gp), that was able to extrudes a variety of antineoplastic drugs from resistant cancer cells, and Carbonic Anhydrase XII (CA XII), that catalyzes the conversion of carbon dioxide to bicarbonate and proton, are overexpressed in some neoplastic cells and are both responsible of the Multidrug Resistance (MDR). New hybrids having inhibitory properties toward both targets were then synthesized. Their structure contains two ester groups so it was important evaluating their stability as a preliminary in vitro tests therefore in this study a series of drug stability experiments was carried out in human plasma (H-pl) and phosphate-buffer solutions (PBS) to assess the possible susceptibility of the ester bonds to plasma enzymes or the spontaneous hydrolysis respectively. For this purpose, a liquid chromatography coupled with a mass spectrometer system, operating in tandem mass spectrometry (LC-MS/MS), was used. For the method a Phenomenex C18 20 mm length, 2 mm internal diameter and 3 microm particle size was used. The solvents used were 10 mM formic acid and 5 mM ammonium formate in water: acetonitrile 90:10(solvent A) and 10 mM formic acid and 5 mM ammonium formate in acetonitrile:water 90:10 (solvent B) according to the elution gradient as follows: initial at 90% solvent A, which was then decreased to 10% in 4.0 min, kept for 2.0 min, returned to initial conditions in 0.1 min and maintained for 2.0 min for reconditioning, to a total run time of 8.0 min. Drug stability experiments relied on incubation of each analyte, in PBS and H-pl, for different times (0, 30, 60 and 120 minutes) and analyzed with the method described before. Also, an evaluation of Matrix Effect following Matuszewski protocol was employed. Furthermore this new hybrids are isomer pairs, so they require long chromatographic analysis for separate them. Our approach, instead, use short columns, fast analysis and LEDA algorithm, a mathematical tool that allows the characterization of coeluting isomers relying on their different MS/MS spectra, increasing the productivity and avoiding any operator error due to contamination.