Validated HPLC Method for Quantification of a Novel Trk Inhibitor, Larotrectinib in Mice Plasma: Application to a
Harsha K Tripathy1, S V Nair Manju1, Ashok Zakkula2
1Karnataka College of Pharmacy Bangalore, Medicinal Chemistry, Bangalore, India.
Abstract:
Larotrectinib, is an orally active novel small molecule approved for the treatment of solid tumors in pediatrics and adult patients. It acts by inhibiting tropomyosin receptor kinase. In this paper, we report the development and validation of a high-performance liquid chromatography (HPLC) method for the quantitation of larotrectinib in mice plasma as per the FDA regulatory guideline. Plasma samples processing was accomplished through simple protein precipitation using acetonitrile enriched with internal standard (IS, enasidenib). The chromatographic analysis was performed using a gradient mobile phase comprising 10 mM ammonium acetate and acetonitrile at a flow-rate of 0.8 mL/min on an X-Terra Phenyl column. The UV detection wave length was set at λmax 262 nm. Larotrectinib and the IS eluted at 3.85 and 6.60 min, respectively with a total run time of 8.0 min. The calibration curve was linear over a concentration range of 0.20-5.00 μg/mL (r2=≥0.992). The intra- and inter-day precision and accuracy results were within the acceptable limits. Results of stability studies indicated that larotrectinib was stable on bench-top, in auto-sampler, up to three freeze/thaw cycles and long-term storage at -80°C. The validated HPLC method was successfully applied to a pharmacokinetic study in mice.
Insights
A new high-performance liquid chromatography (HPLC) method accurately quantifies larotrectinib in mouse plasma. This validated method supports pharmacokinetic studies for this important cancer therapeutic.
Area of Science:
- Pharmacology
- Analytical Chemistry
- Biochemistry
Background:
- Larotrectinib is an orally active small molecule inhibitor of tropomyosin receptor kinase (TRK) used to treat solid tumors.
- Accurate quantification of larotrectinib in biological matrices is essential for pharmacokinetic (PK) studies.
- Existing methods may require improvement in terms of sensitivity, efficiency, or regulatory compliance.
Purpose of the Study:
- To develop and validate a high-performance liquid chromatography (HPLC) method for quantifying larotrectinib in mice plasma.
- To ensure the method adheres to US Food and Drug Administration (FDA) regulatory guidelines.
- To apply the validated method in a mouse PK study.
Main Methods:
- Plasma samples were prepared using protein precipitation with acetonitrile and an internal standard (enasidenib).
- Chromatographic separation was achieved using a gradient mobile phase (ammonium acetate and acetonitrile) on an X-Terra Phenyl column.
- UV detection was performed at 262 nm, with a total run time of 8 minutes.
Main Results:
- The HPLC method demonstrated linearity over a concentration range of 0.20-5.00 μg/mL (r² ≥ 0.992).
- Intra- and inter-day precision and accuracy were within acceptable regulatory limits.
- Larotrectinib stability was confirmed under various conditions, including bench-top, auto-sampler, freeze/thaw cycles, and long-term storage at -80°C.
Conclusions:
- A robust and validated HPLC method for larotrectinib quantification in mouse plasma was successfully developed.
- The method is suitable for supporting PK studies in preclinical research.
- This validated assay provides a reliable tool for assessing larotrectinib pharmacokinetics.
