Combination of micellar electrokinetic and high-performance liquid chromatographies to assess age-related changes in the in vitro metabolism of Fischer 344 rat liver.

The metabolism of doxorubicin, a widely used anticancer drug, is different in young adult and old cancer patients. In this study, we demonstrate that micellar electrokinetic chromatography with laser-induced fluorescence detection is highly suited to monitor the metabolism of doxorubicin in subcellular fractions isolated from young adult (11 months, 100% survival rate) and old (26 months, ~25% survival rate) Fischer 344 rat livers. The relative amounts of doxorubicin metabolized in both mitochondria-enriched and postmitochondria fractions of young adult were larger than the respective fractions of old rat liver. 7-Deoxydoxorubicinolone and 7-deoxydoxorubicinone were identified using internal standard addition and structural elucidation by high-performance liquid chromatography with combined laser-induced fluorescence and mass spectrometry detection. Although high-performance liquid chromatography with combined laser-induced fluorescence and mass spectrometry detection is more useful in the identification of compounds, micellar electrokinetic chromatography with laser-induced fluorescence detection has low-sample requirements, simplified sample processing procedures, short analysis times and low limit of detection. Therefore, the combination of these two techniques provides a powerful approach to investigate metabolism of fluorescent drugs in aging studies.

Tandem laser-induced fluorescence and mass spectrometry detection for high-performance liquid chromatography analysis of the in vitro metabolism of doxorubicin.

Structural characterization, identification, and quantification of xenobiotics and their metabolic products commonly require the use of at least two different techniques. This has been the case in the analysis of metabolic products of doxorubicin, a widely used fluorescent anthracycline for the treatment of tumors and leukemia. In this work, we combine high-performance liquid chromatography (HPLC) with a tandem laser-induced fluorescence (LIF) and mass spectrometry (MS) detection scheme for the characterization of doxorubicin and its metabolites produced in the postmitochondrial fraction prepared from Fischer 344 rat liver. LIF detection allowed quantification of the metabolic compounds while MS detection aided in the identification of the metabolites. Using this HPLC-LIF-MS methodology, the disappearance of doxorubicin and the appearance of 7-deoxydoxorubicinone and 7-deoxydoxorubicinolone were monitored over the course of 40 min. This application demonstrates the potential of the tandem LIF-MS detection scheme in quantification and characterization of biotransformations of fluorescent xenobiotics of biomedical and environmental relevance. Furthermore, this detection scheme would be particularly relevant in the analysis of fluorescent analytes in complex samples and in validation of methods for the analysis of such samples that typically rely only on LIF detectors.