Comparative Study of Various Procedures for Extracting Doxorubicin from Animal Tissue Samples

This article presents a comparative study of selected deproteinization-, liquid–liquid-extraction- (LLE), and solid-phase-extraction (SPE)-based procedures for the isolation of doxorubicin (DOX) and daunorubicin (DAU) as an internal standard (IS) from rat tissue samples. During the experiments, all samples were analyzed via liquid chromatography coupled with fluorescence detection (LC-FL), with analytes being monitored at excitation and emission wavelengths of 487 and 555 nm, respectively. The absolute recoveries of the sample-preparation procedure were then calculated and compared, and the advantages and disadvantages of each approach were considered in depth. Ultimately, SPE with hydrophilic–lipophilic balanced (HLB) sorbents was selected as the most effective extraction procedure as it enabled the absolute recovery of DOX from tissue samples at a level of 91.6 ± 5.1%. Next, the selected HLB-SPE protocol was coupled with LC-FL separation and the resultant method was validated according to FDA and ICH requirements. The validation data confirmed that the developed procedure met all required criteria for bioanalytical methods, with a limit of detection (LOD) and limit of quantification (LOQ) of 0.005 µg/g and 0.01 µg/g, respectively. Finally, the developed protocol was successfully tested on various rat tissues enriched with DOX, confirming its potential as an interesting alternative to previously reported protocols for pharmacokinetic studies and clinical investigations aimed at analysis of the level and biodistribution of DOX in tissue samples after systemic administration of this drug.