Quantitative subcellular study of doxorubicin in MCF-7/Adr cells using liquid chromatography–tandem mass spectrometry

• Confocal microscopy is the common method to determine doxorubicin in subcellular level, but it has a complex operation and cannot quantitative determination.
• We quantitative doxorubicin in subcellular level by LC/MS/MS.
• This method is rapid, sensitive and selective.

A rapid, sensitive and selective high-performance liquid chromatography–tandem mass spectrometric (LC–MS/MS) method has been developed and validated for the determination of doxorubicin in intracellular compartments using glibenclamide as internal standard (IS). MCF-7/Adr cancer cells (1 × 106) were incubated with doxorubicin (8 μg/mL) for 0.5, 1, 2 and 4 h and then subjected to sequential extraction of cytosolic, membrane/organelle, nuclear and cytoskeleton soluble protein. Samples were extracted using protein precipitation with methanol. Chromatographic separation was carried out on a C18 column with acetonitrile and 0.1% formic acid water as mobile phase and with gradient elution at a flow rate of 0.2 mL/min. The method was linear over the range of 1–300 ng/mL with a lower limit of quantification (LLOQ) of 1 ng/mL. The distribution of doxorubicin in subcellular components of MCF-7/Adr cancer cells was mainly in nucleic protein fraction.