Abstract

Tucatinib, a selective HER2 tyrosine kinase inhibitor demonstrating unique survival benefits for HER2-positive breast cancer patients, can be precisely measured in rat plasma using a validated LC-MS/MS method. To ensure reliable quantification, the method incorporates Imatinib as an internal standard (IS). This allows for the accurate measurement of Tucatinib concentrations over a wide linear range, spanning from 0.05 to 1000 ng/ml. Protein precipitation is employed for sample preparation. This involves adding 2:1 acetonitrile (200 μl) to rat plasma (100 μl) to extract both Tucatinib and the IS. The combination of a Kinetex C18 column and a gradient mobile phase system achieves rapid and clean separation of Tucatinib and the IS in only 1.5 minutes. For separation, two mobile phases: A (water with 0.1% formic acid) and B (a 50:50 mix of acetonitrile and methanol, both with 0.1% formic acid) were used. This approach facilitated the sensitive detection of both Tucatinib and the IS using a specific method named positive electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. ESI-MRM enhances the selectivity of the analysis, ensuring we only measure the desired compounds. The method achieves a low limit of quantification (LLOQ) of 0.05 ng/ml, indicating its high sensitivity for detecting Tucatinib. Excellent linearity was observed for the standard curve (r² > 0.9997) across a broad concentration range of 0.05 to 1000 ng/ml. This signifies a strong, positive relationship between Tucatinib concentration and the instrument›s response. Furthermore, the method adheres to the established criteria set by the Food and Drug Administration for both precision and accuracy, assessed within and between days (intra- and inter-day). To ensure the method’s robustness, stability studies evaluated Tucatinib and the IS under various conditions, including bench-top stability, auto-sampler compatibility, long-term storage, and freeze-thaw cycles. This successful validation paves the way for the method’s reliable application in pharmacokinetic studies.

Key words: Tucatinib, LC-MS/MS, method validation, Positive electrospray ionization, Pharmacokinetics