Challenging core-shell stationary phases with the separation of closely related anti-cancer compounds: performance studies and application to drug quantitation in cell cultures with multi-well plate clean-up

• Fully-porous and core-shell columns with three functionalizations were compared.
• An optimized system with a conventional-cell fluorescence detector was employed.
• Core-shell columns immediately improved the performance of the separation.
• An analytical application for cell culture samples with 96-well plate clean-up was fully validated.
• The method notably reduced the volume of organic solvent compared to a previous one.

In the present paper, we compare the chromatographic behaviour of six different stationary phases (5 μm fully porous and 2.6 μm core-shell particles, featuring octadecyl-, pentafluorophenyl-propyl- and phenyl-hexyl- functionalizations) for the separation of camptothecin and a family of closely related analogues of the natural anti-cancer drug luotonin A under different experimental conditions (organic solvent nature and proportion, temperature and presence of modifiers). We found two differentiated behaviours for the phenyl-hexyl-functionalized supports depending on the temperature. The efficiency and kinetic performance of fully porous vs. core-shell particles was compared by means of van Deemter and kinetic plots, using an optimized conventional HPLC system. Columns packing core-shell particles afforded improved efficiencies, permeabilities, analysis times and consumption of solvents. Nevertheless, our optimized system was not able to release the full intrinsic efficiencies of these columns. The replacement of acetonitrile with methanol had a dramatic effect in the behaviour of the pentafluorophenyl-propyl stationary phase. The aggregate of these results led us to select the core-shell C-18 stationary phase along with a H2O:MeCN gradient for the rapid and sustainable quantitation of the anti-tumour agents in cell cultures. A simple and rapid clean-up step was optimized using 96-well deproteinization and delipidation plates. The validation of the proposed analytical method showed excellent figures of merit. The combination of this sample pre-treatment procedure with the separation on core-shell particles yielded a fast methodology able to process a large number of samples with minimal waste generation and environmental impact.