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Micellar electrokinetic chromatography coupled with tandem mass spectrometry (MEKC-MS/MS) is a powerful technique for various applications due to its ability to analyze a wide range of compounds in a highly efficient and environmentally friendly manner. On the other hand, there is an unmet need for therapeutic drug monitoring of breast cancer drugs. There are some LC-MS/MS methods reported for the simultaneous determination of abemaciclib (ABE), ribociclib (RIB), palbociclib (PAL), anastrozole (ANA), letrozole (LET) and fulvestrant (FUL) [1-2]. However, here we report an optimization of separation for these drugs' first- ever MEKC-MS/MS method. Separation was obtained using a volatile, MS-compatible surfactant ammonium perfluorooctanoate (APFO). Optimization of separation was performed by testing APFO concentration (75-150 mM), pH of the running buffer (9.0-10.5), a fraction (20-40 %) and type (MeOH, EtOH, AcN, i-PrOH, THF) of organic modifiers in the running buffer, voltage (20-25 kV), capillary temperature (20-30 °C), duration of hydrodynamic injection (5-50 s) and hydrodynamic pressure during the analysis (0-100 mbar). Higher APFO concentrations yielded better resolution until 125 mM after which resolution worsened and the capillary current exceeded the recommended 50 µA. With the increase of pH of the running buffer separation improved. Among the organic modifiers, MeOH was shown to be the best for both separation and peaks’ shapes and symmetries. The addition of other organic modifiers resulted in deteriorated separation, possibly due to the interference with micelle formation. Higher hydrodynamic pressure during the analysis shortened each run without the loss of resolution. Voltage and capillary temperature did not affect separation significantly so starting values were chosen as a compromise between run time and capillary current. Optimal separation conditions were thus shown to be 125 mM APFO in 32 % methanol with an apparent pH of 10.5, a capillary temperature of 30 °C, separation voltage of 25 kV, hydrodynamic injection of 30 s under 50 mbar and hydrodynamic pressure during the analysis of 100 mbar. Additionally, the sweeping on-line preconcentration of analytes was performed by dissolving the analytes in 32% MeOH, but without the micellar pseudo-stationary phase. With this approach we have achieved that the negatively charged micelles from the running buffer penetrate the sample zone and “sweep” the analytes, thus concentrating them in a narrow band inside the capillary. In further studies, the MS ion source will be optimized for this method and the method will be validated for its intended purpose. This work has been fully supported by the Croatian Science Foundation through projects UIP-2019-04- 8461 and DOK-2021-02-4595, and the European Regional Development Fund, project Farminova, KK.01.1.1.02.0021. |
