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

•An UPLC-MS/MS method is developed for simultaneous determination disulfide-linked doxorubicin prodrug and activated doxorubicin in cancer cells.•The method provides efficient sample preparation with acetonitrile precipitation and remove of intercalated doxorubicin through bath sonication.•The method is suitable for the in vitro study of doxorubicin prodrug nanoparticles.

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 (r2) ≥ 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 (<15%) and relative error (RE%) for accuracy was between −5.35 and 9.18% for all analytes. Recovery (59.28-69.53% for DOX-S-S-DOX and 99.13-100.10% for DOX) and matrix effect (99.69-111.19%) was consistent, precise, and reproducible at different quality control levels in accordance with FDA guidance. Stability studies showed that DOX-S-S-DOX was unstable both during the bench-top and long-term storage, while the stability during sample preparation and LC-MS runtime was suitable for all the analytes. Hence, the samples should be prepared as soon as possible at the time point to prevent the catabolism of DOX-S-S-DOX. The assay was successfully used in the cellular metabolism and pharmacokinetics study of DOX-S-S-DOX and it may give a clue to explore analytical methods of other prodrug forms of DOX.

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