title: Ultra-sensitive assay for paclitaxel in intracellular compartments of A549 cells using liquid chromatography-tandem mass spectrometry.
abstract: 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 (10(6)) were incubated with paclitaxel (2ng/mL) for up to 4h 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 (50mm×4.6mm, 2.7μm) 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 2-600pg/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.
authors.affiliation: Clinical Pharmacology Center, Research Institute of Translational Medicine, The First Bethune Hospital of Jilin University, Dongminzhu Street, Changchun 130061, PR China.
authors.forename: Tingting
authors.initials: T
authors.lastname: Wang
authors.forename: Wenxiao
authors.initials: W
authors.lastname: Ma
authors.forename: Yantong
authors.initials: Y
authors.lastname: Sun
authors.forename: Yan
authors.initials: Y
authors.lastname: Yang
authors.forename: Weiping
authors.initials: W
authors.lastname: Zhang
authors.forename: J Paul
authors.initials: JP
authors.lastname: Fawcett
authors.forename: Hongwei
authors.initials: H
authors.lastname: Du
authors.forename: Jingkai
authors.initials: J
authors.lastname: Gu
mesh_terms: D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D059625:Liquid-Liquid Extraction / Q000379:methods*; D017239:Paclitaxel / Q000032:analysis*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D017239:Paclitaxel