A sensitive liquid chromatography/electrospray tandem mass spectroscopy method for simultaneous quantification of a disulfide bond doxorubicin conjugation prodrug and activated doxorubicin: Application to cellular pharmacokinetic and catabolism studies

Abstract In recent years, drug conjugates as a prodrug strategy have been widely studied, especially combined with nanotechnology. Disulfide-linked doxorubicin drug–drug conjugate (DOX-S-S-DOX) nanoparticles, have recently been developed as a doxorubicin prodrug nanoparticles with greater anticancer activity and less toxicity than doxorubicin in vivo , while its intracellular kinetics and metabolism is unclear which may provide us with a deeper understanding of its pharmacological mechanism and antitumor effect. Hence, in this study, a rapid and sensitive ultra high-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method was developed to detect doxorubicin (DOX) activated from DOX-S-S-DOX, as well as the prodrug itself in human breast cancer tumor cells (MCF-7). Sample preparation involved acetonitrile precipitation to extract the analytes simultaneously and bath sonication to remove intercalated DOX from DNA. The calibration range was 3–60 ng/mL for DOX and 20–400 ng/mL for DOX-S-S-DOX with the correlation coefficients (r 2 ) ≥ 0.99, using daunorubicin as internal standard (IS). The inter- and intra-assay precision (relative standard deviation, RSD%) of quality control samples was in the acceptable range (