engleski

Optimisation studies of solid phase extraction and LC-MS analysis of CDK4/6 inhibitors in breast cancer combination therapy in plasma samples

Palbociclib, ribociclib and abemaciclib are novel anticancer agents used for HR+, HER2- breast cancer treatment in combination with anastrozole, letrozole or fulvestrant. They are prone to inter- individual variabilities and concentration- dependent adverse events, therefore better treatment outcomes might be achievable with therapeutic drug monitoring (TDM). One of the main prerequisites to TDM is the existence of a reliable and, preferably, fast, simple, and ecologically acceptable bioanalytical method. Few bioanalytical methods have been published so far for the determination of all these six drugs in plasma, however using only protein precipitation for sample clean-up. In this work, a solid-phase extraction (SPE) procedure was optimised for the simultaneous extraction of all six drugs of interest from plasma. Several SPE phases were tested: octylsilyl (C8, 200 mg/3 mL, 500 mg/3 mL), octadecylsilyl (C18, 200 mg/3 mL), hydrophilic- lipophilic balance (HLB, 60 mg/3 mL), mixed-mode cation exchange (MCX, 30 mg/1 mL), and mixed-mode weak cation exchange (WCX, 60 mg/3 mL). Analytes were eluted with methanol, acetonitrile, 2 % formic acid in methanol or 5 % ammonia in methanol. The samples were analysed with Agilent 1290 Infinity II ultra-high performance liquid chromatograph coupled to Agilent 6470 triple quadrupole mass spectrometer. Waters XBridge Phenyl column (150 × 4.6 mm, 2.5 μm) was used as the stationary phase at 35 °C. The mobile phase consisted of water and methanol with 0.1 % formic acid in gradient elution at a 0.6 mL/min flow rate. The optimal solution was found to be the C8 phase (200 mg/3 mL), eluted with 1500 µL of methanol, which yielded extraction recoveries above 90 % for all analytes. This method was validated in terms of intra and inter-day precision and accuracy, linearity and calibration in the clinically relevant ranges, selectivity, carry-over, and matrix effects. The validated method was then successfully applied for the analysis of samples from patients taking different combinations of the drugs of interest. The proposed method is simple, fast, sensitive, providing good sample clean-up and high extraction yields for all six analytes of interest. Its application on real patient plasma samples supports its suitability for the purposes of further TDM testing. ACKNOWLEDGEMENTS This work has been fully supported by the Croatian Science Foundation, under the project number UIP-2019-04- 8461 and DOK-2021-02-4595.

CDK4/6 inhibitors ; solid phase extraction ; plasma

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