Optimized LC-MS/MS method for Doxorubicin quantification: validating drug uptake and tumor reduction in zebrafish xenograft model of breast cancer

Doxorubicin, a potent chemotherapeutic drug, is widely used against various cancers, notably breast cancer. While its efficacy is well-documented, precise dosage determination in experimental models remains challenging. Zebrafish xenografts of various cancers confirm doxorubicin's anti-cancerous effect; however, since doxorubicin treatment of zebrafish larva is done by adding doxorubicin to fish water, the precise chemotherapeutic dosage for zebrafish larva remains unknown. In this study, we provide a liquid chromatography tandem mass-spectrometry (LC-MS/MS) method for quantifying doxorubicin uptake in zebrafish larvae and thus provide a direct estimate of doses required for the therapeutic effect. Alongside quantification, we measured the therapeutic effect of doxorubicin in zebrafish larvae xenografted with triple negative breast cancer cell line, MDA-MB-231. LD50 value of doxorubicin was first determined by incubating 3-days post fertilization (dpf) larvae with different doses of doxorubicin for 72 h. Doxorubicin was quantified both from zebrafish larval homogenate and treatment solution. Analysis was performed by selected-reaction monitoring (SRM) scans in positive ionization mode. LD50 value for 72 h calculated to be 35.95 mg/L. As expected, doxorubicin-treated xenografts exhibited a significant reduction in tumor growth. The range of limit of detection (LOD) and limit of quantification (LOQ) for doxorubicin were 2 and 5 μg/L respectively. Intra- and inter-day accuracy was within the range of 82-114%. Overall, in this study we describe a reliable method for quantifying doxorubicin in zebrafish larvae. Our study facilitates precise dosage estimation, enhancing the relevance of zebrafish xenograft model in cancer research and potentially improving translational applications of chemotherapeutic treatments.