Development and validation of an LC-MS/MS method for monitoring larotrectinib, a tropomyosin-related kinase inhibitor, in mouse and human plasma and application to pharmacokinetic studies

Abstract

Larotrectinib is an orally administered drug and the first and only selective pan-tropomyosin receptor kinase (TRK) inhibitor in clinical development to treat cancer patients harboring a neurotrophic receptor tyrosine kinase gene fusion. In this study, an analytical method to quantify the TRK inhibitor in mouse and human plasma was developed and validated using LC-MS/MS following protein precipitation with acetonitrile. Larotrectinib and an internal standard (carbamazepine) were separated from endogenous substances using an Xterra C18 column with acetonitrile containing 0.1% formic acid as the mobile phase. The ions m/z 429.8 → 342.8 for larotrectinib and m/z 237.0 → 194.0 for carbamazepine detected in multiple reaction monitoring mode were used for the quantitation. The detector response of larotrectinib was linear within the concentration range 5–10,000 ng/mL with a correlation coefficient (r2) of not less than 0.999. The intra- and inter-day precision and accuracy were less than 10.48% and within − 8.99%, respectively, in mouse and human plasma. Larotrectinib was stable under various storage and handling conditions, and no significant matrix effect was observed in both mouse and human plasma. Finally, the assay was successfully applied to the pharmacokinetic study of larotrectinib after its intravenous and oral administration to mice.

A dried blood spot assay with HPLC–MS/MS for the determination of larotrectinib in mouse blood and its application to a pharmacokinetic study

“…Having front end liquid chromatography as a UPLC with a diverter valve and the suggested automation is not feasible in most research laboratories and academic institutes. However, in the other two reported methods (Attwa et al, 2020; Chae et al, 2020) the authors have used regular HPLC as a front end to the mass spectrometer. Elution was done using either a linear gradient (Chae et al, 2020) or isocratically (Attwa et al, 2020).…”

“…To date, few bioanalytical methods have been reported for the quantification of larotrectinib in biological matrix (Attwa, Kadi, & Darwish, 2020; Chae, Song, Chae, et al, 2020; Sparidans, Wang, Schinkel, Schellens, & Beijnen, 2018; Tripathy et al, 2020). Sparidans et al (2018) reported the first bioanalytical method for the quantification of larotrectinib from mouse plasma and tissues using LC–MS/MS.…”

“…The linearity range was 1–2,000 ng/ml (Sparidans et al, 2018). The LC–MS/MS method reported by Chae et al (2020) was validated in both human and mouse plasma. The validated method was applied in a mouse pharmacokinetic study.…”

“…Larotrectinib and the IS (carbamazepine) were separated on an Xterra C 18 column using linear gradient elution with a flow‐rate of 0.4 ml/min. The linearity range was 5–10,000 ng/ml (Chae et al, 2020). Attwa et al (2020) reported an LC–MS/MS method to support the metabolic stability assessment of larotrectinib in human liver microsomes.…”

“…The flow‐rate was 0.2 ml/min (Attwa et al, 2020). In all of the reported LC–MS/MS methods, larotrectinib was quantified using the transition pair (Q1 → Q3) of m/z 429 → 342 (Attwa et al, 2020; Chae et al, 2020; Sparidans et al, 2018). Very recently, Tripathy et al (2020) reported an HPLC method for the quantitation of larotrectinib from mouse plasma.…”