Bioanalysis of ibrutinib, and its dihydrodiol- and glutathione cycle metabolites by liquid chromatography-tandem mass spectrometry

Abstract

Ibrutinib is a targeted covalent inhibitor frequently used for the treatment of various lymphomas. In addition to oxidative metabolism, it is metabolized through glutathione coupling. The quantitative insight into this kind of metabolism is scarce, and tools for quantitation are lacking. The non-oxidative metabolism could prove a more prominent role when oxidative metabolism is impaired. Also, in-vitro studies could over-estimate the effect of CYP450-inhibition. To gain quantitative insight into this relatively unknown biotransformation pathway of the drug we have developed a validated simple, fast and sensitive bio-analytical assay for ibrutinib, dihydrodiol-ibrutinib, and the glutathione, cysteinylglycine and cysteine conjugates of ibrutinib in human plasma. The method emphasizes on simplicity, the thiol-conjugates were synthesized by a simple one step synthesis, followed by LC-purification. Sample preparation was done by a simple protein crash with acetonitrile containing labeled internal standards, evaporation of solvents, and reconstitution in eluent. Finally, the compounds were quantified using UHPLC-MS/MS. The assay was successfully validated in a 0.5-500nM calibration range for all compounds, and also a lower range of 0.05-50 nM was demonstrated for ibrutinib to accommodate for even the lowest trough levels. This assay has a considerably higher sensitivity than previous published assays, with the previous lowest LLOQ being 1.14 nM. Both, ibrutinib, dihydrodiol-ibrutinib and the cysteine conjugate were deemed stable under refrigerated or frozen storage conditions. At room temperature, the glutathione conjugate showed rapid degradation into the cysteinylglycine conjugate in plasma. Finally, the applicability of the assay was demonstrated in patient samples.

In vitro metabolism of ibrutinib in rat, dog and human hepatocytes using liquid chromatography combined with diode‐array detection and Q‐Exactive Orbitrap tandem mass spectrometry

“…To the best of our knowledge, there are several publications with regard to the pharmacokinetics of ibrutinib . Ibrutinib was rapidly absorbed into the body after oral administration and reached its maximum plasma concentration at 1–2 h post‐dose with an elimination half‐life of 4–6 h .…”

“…[9][10][11] Ibrutinib was rapidly absorbed into the body after oral administration and reached its maximum plasma concentration at 1-2 h post-dose with an elimination half-life of 4-6 h. 8 The primary metabolite of ibrutinib was dihydrodiolibrutinib. [9][10][11] This metabolite showed reversible inhibitory activity towards BTK, approximately 15 times lower than that of ibrutinib with plasma concentrations in the same range as ibrutinib levels. 4,12 In vitro experiments indicated that ibrutinib was extensively metabolized primarily by CYP3A4 and to a lesser extent by CYP2D6.…”

The effect of naringenin on the pharmacokinetics of ibrutinib in rat: A drug–drug interaction study

“…There have been several reports on the LC/MS analysis of ibrutinib in plasma (Beauvais et al, ; de Vries et al, ; Rood et al, ). All of the assays were based on the triple quadrupole mass spectrometer with selective reaction monitoring mode, which showed high sensitivity and selectivity.…”

A review of bioanalytical methods for chronic lymphocytic leukemia drugs and metabolites in biological matrices

“…The application of the method is not shown (Huynh et al, ). Rood et al () demonstrated the bioanalysis of ibrutinib, its dihydrodiol and glutathione metabolites in human plasma using LC‐ESI‐MS/MS. The chromatographic separation was performed on a Waters Acquity UPLC BEH C18 column using 0.1% formic acid and methanol as the mobile phase.…”