Effective quantification of 11 tyrosine kinase inhibitors and caffeine in human plasma by validated LC-MS/MS method with potent phospholipids clean-up procedure. Application to therapeutic drug monitoring

Therapeutic drug monitoring (TDM) help to improve treatment efficacy and safety. Therefore, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous monitoring of 11 tyrosine kinase inhibitors (TKIs) in human plasma. This included in the assay are used in the treatment of chronic myeloid leukemia (CML: imatinib, dasatinib, nilotinib, bosutinib, ponatinib), polycythemia vera (ruxolitinib), chronic lymphocytic leukemia (ibrutinib) and rheumatoid arthritis (filgotinib, tofacitinib, baricitinib, peficitinib). Caffeine was also included in the method. Caffeine increases the acidity of the stomach and decreases its pH as well as is a competitive inhibitor of cytochrome P450 isoenzymes. Thus, it may influence absorption and metabolism of some TKIs, by modifying their plasma levels. The analytes of interest and their stable isotope-labeled internal standards were extracted from 200 mu L of human plasma. Microelution-solid phase extraction (mu-SPE) was optimized for method validation and compared to simple protein precipitation (PPT). A gradient elution on a Poroshell 120 EC-C18 column at 60 degrees C and a flow rate of 0.5 mL/min was applied for analyte separation. The analytical run lasted 8 min and it was followed by a reequilibration time of 4 min. Dynamic multiple reaction monitoring scan in the positive ionization mode was applied to improve method sensitivity. Endogenous plasma phospholipids can strongly affect MS analysis. Hence, the monitoring of endogenous phospholipids was included in the assay. Full validation of the method was achieved, including tests of precision, accuracy, trueness, linearity, extraction recovery, matrix effect, process efficiency, stability, sensitivity (with excellent LLOQs), selectivity, identity confirmation and carry-over effect. Regarding sample cleanup, more than 91% of early eluting and more than 96% of late eluting endogenous phospholipids were eliminated by mu-SPE when compared to PPT. This method enables the simultaneous plasma monitoring of 11 TKIs and caffeine and ensures high effectiveness in phospholipids elimination. The present approach is currently used in our clinical practice, being applied to TDM of dasatinib, imatinib, nilotinib and ponatinib. TKIs plasma monitoring helps to individualize dose adjustment and manage adverse effects in CML patients.