Ultra-sensitive assay for paclitaxel in intracellular compartments of A549 cells using liquid chromatography–tandem mass spectrometry

A high-performance liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for the determination of paclitaxel in intracellular compartments using docetaxel as internal standard (IS) has been developed and validated. A549 cancer cells (106) were incubated with paclitaxel (2 ng/mL) for up to 4 h and then subjected to sequential extraction of cytosolic, membrane/organelle, nuclear and cytoskeleton soluble protein. Fractions were ultrasonicated to release protein bound paclitaxel after which drug was extracted using liquid–liquid extraction with diethyl ether:dichloromethane (2:1, v/v). Chromatographic separation was then carried out on an Ascentis Express C18 column (50 mm × 4.6 mm, 2.7 μm) with a mobile phase of acetonitrile:0.1% formic acid in water (50:50, v/v). Detection involved electrospray positive ionization followed by multiple reactions monitoring of the precursor-to-product ion transitions of paclitaxel at m/z 854.4 → 286.3 and docetaxel at m/z 808.6 → 226.1. Assay validation based on samples of total cell extract in the same buffer as protein fractions showed the assay was linear over the range 2–600 pg/mL with intra- and inter-day precision (as relative standard deviation) and accuracy (as relative error) of <7% and <±12%, respectively. Recovery was approximately 70% and matrix effects were minimal. The distribution of paclitaxel in subcellular components of A549 cancer cells was mainly into the cytoskeletal compartment.

► The LLOQ of this assay is 2 pg/mL, which is lower than ever before (20 pg/mL).
► The common method to determine the anticancer drugs binding to microtubules is radioactive analysis.
► Radioactive analysis could affect the normal physiological state of cells and could not differentiate drug and metabolites.
► This method is simple and reproducible, diminit matrix interferences.