An LC-MS/MS method for the quantification of doxorubicin uptake in zebrafish larvae breast cancer xenografts

Doxorubicin, a potent chemotherapeutic drug, is widely used against various cancers, notably breast cancer. Studies with zebrafish xenografts of different cancers treated with doxorubicin indicated that doxorubicin is effective against tumors. However, because treatment in zebrafish larva is usually done by adding doxorubicin to the fish water, the precise chemotherapeutic dosage for zebrafish larva remains unknown. This study presents a liquid chromatography tandem mass-spectrometry (LC-MS/MS) method to quantify doxorubicin uptake in zebrafish larvae, enabling accurate dose estimation for tumor reduction in triple negative breast cancer (MDA-MB-231) xenografts. We first determined doxorubicin toxicity in zebrafish embryos by adding various drug concentrations to the fish water, identifying sublethal doses based on survival and development endpoints. MDA-MB-231 zebrafish xenografts were subsequently treated with a sub-lethal dose of doxorubicin. Doxorubicin uptake in zebrafish larvae was measured by LC-MS/MS with the limit of detection and lower limit of quantification for doxorubicin as 2 and 5 μg/L respectively. Precent accuracy was within the range of 82-114 %. Our developed and validated LC-MS/MS method consistently meets international standards. Despite low uptake (2.06 ± 1.01 ng/larva, calculated value below the lower limit of quantification) after three days, zebrafish breast cancer xenografts showed tumor reduction indicating therapeutic effects of doxorubicin. Unlike earlier phenotypic-based methods, our approach quantifies doxorubicin levels in-vivo in zebrafish xenograft model, allowing precise correlation between drug exposure and efficacy.