DEVELOPMENT AND VALIDATION OF A SENSITIVE LC-MS/MS ANALYTICAL METHOD FOR QUANTIFYING AXITINIB WITH AXITINIB D3 AS AN INTERNAL STANDARD:

KUPPIRALA SANDEEP CHAKRAVARTHI; KANALA SOMASEKHAR REDDY

doi:10.22159/ajpcr.2025v18i10.54999

Authors

KUPPIRALA SANDEEP CHAKRAVARTHI Department of Pharmaceutical Sciences, Jawaharlal Nehru Technological University Anantapur, Andhra Pradesh, India https://orcid.org/0009-0005-7568-2856
KANALA SOMASEKHAR REDDY Department of Pharmacology, Raghavendra Institute of Pharmaceutical Education and Research - Autonomous, Ananthapuramu, Andhra Pradesh, India. https://orcid.org/0000-0002-9206-900X

DOI:

https://doi.org/10.22159/ajpcr.2025v18i10.54999

Keywords:

Axitinib, Axitinib D3,, Food and drug administration, European medicines agency, Pharmacokinetics

Abstract

Objective: An efficient, highly sensitive, and robust LC-MS/MS method has been optimized, validated for accurately measuring Axitinib concentrations in human plasma, utilizing Axitinib D3 as an internal standard.

Methods: A liquid–liquid extraction approach was utilized to isolate both the analyte and the internal standard from the plasma matrix. An Eclipse Phenyl column of 100 × 3.0 mm size with 5 µm particles operated using a mobile phase containing methanol mixed with 0.1% formic acid in 10 mM ammonium formate at a ratio of 60:40 (v/v) following the advanced bioanalytical framework for method establishment and validation.

Results: The complete chromatographic run was accomplished in 4.0 minutes, with the analyte and internal standard eluting at approximately 1.89 minutes. The assay demonstrated a validated linear response across the concentration range of ~ 0.2 to 125 ng/mL. The developed method was thoroughly validated for key parameters including precision, accuracy, selectivity, matrix effects, and stability. All validation results met the predefined specifications, confirming method's reliability.

Conclusion: The validated method for quantifying Axitinib in human plasma complies with regulatory guidelines set by the FDA and EMA, making it appropriate for pharmacokinetic studies in human subjects.

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