Simultaneous quantitative determination of arachidonic acid and cascade metabolites in rat serum by UPLC-MS/MS: application for longitudinal metabolomics of anlotinib

Abstract

Arachidonic acid (AA) and cascade metabolites have been shown to be involved in cancer pathologic states. Anlotinib, a novel oral small molecule inhibitor of multiple receptor tyrosine kinases, has brought significant improvement to the survival of patients with advanced lung cancer. Here, a robust and reproducible ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed, optimized and validated for quantitating AA and cascade metabolites for the first time. Through careful optimization of the analytical conditions, a total of 69 analytes can be efficiently separated and quantitated in a single run of 17 min. A simple and labor-saving protein precipitation procedure was utilized for serum sample pretreatment. The validation parameters and quality control chart of all analytes satisfy the acceptance criteria of bioanalytical method guidelines. The limit of detection (LOD) ranged from 0.005 ng mL⁻¹ to 1 ng mL⁻¹, and the volume of serum was only 20 μL. This rapid and sensitive UPLC-MS/MS method was successfully applied to a longitudinal metabolomics study in rat serum after a single administration of anlotinib (6 mg kg⁻¹). Finally, a total of 41 metabolites can be calculated under the present conditions. Serum samples from the same rat were segregated into a tight cluster in both unsupervised principal component analysis (PCA) and supervised orthogonal partial least-squares discriminant analysis (OPLS-DA) at different sampling time points after anlotinib treatment. Moreover, the number of analytes whose variable importance (VIP) values were larger than 1.0 was 17. The present study not only offers a UPLC-MS/MS analytical reference for AA but also brings out insights for future mechanistic studies or biomarkers to predict the efficacy, toxicity and clinical outcomes in patients with cancer.