Studies On Pharmacokinetics Of Paclitaxel In Intracellular Compartments And Mass Spectrometric Fragmentation Patterns Of Taxane Drugs

This paper focused on the intracellular pharmacokinetics properties of paclitaxelin the compartments of A549cells, including the dissociation pathways of two taxoldrugs by use of electrospray ion trap mass spectrometry. The result provided areference for investigating the efficacy of different formulations of paclitaxel, andwere applicable to qualitative and quantitative analysis of paclitaxel in vitro and invivo.A high-performance liquid chromatography–tandem mass spectrometric(LC-MS/MS) method for the determination of paclitaxel in intracellularcompartments using docetaxel as internal standard (IS) has been developed andvalidated. A549cancer cells (106) were incubated with paclitaxel (2ng/mL) fordifferent times and then subjected to sequential extraction of cytosolic,membrane/organelle, nuclear and cytoskeleton soluble protein. Fractions wereultrasonicated to release protein bound paclitaxel after which drug was extractedusing liquid–liquid extraction with diethyl ether:dichloromethane (2:1, v/v).Chromatographic separation was then carried out on an Ascentis Express C18column(50×4.6mm,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 multiplereaction monitoring of the precursor-to-product ion transitions of paclitaxel at m/z854.4→286.3and docetaxel at m/z808.6→226.1. The assay was linear over the range2–600pg/mL with intra-and inter-day precision (as relative standard deviation) andaccuracy (as relative error) of <7%and <±12%, respectively. Recovery wasapproximately70%and matrix effects were96%. Assay of the four subcellularfractions showed that the distribution of paclitaxel was dependent on the incubation time. After incubation for0.5h, paclitaxel was mainly present in the cytosol, nuclearand cell membrane whereas after2.5h, it was mainly localized in the cytoskeletalcompartment.The fragmentation pathways of two taxanes drugs have been studied in positiveion mode by Triple TOF with the advantages of high mass accuracy and highresolution analysis. The [M+H]~+ions were observed by ESI-MS, from which themolecular weights were obtained. The accurate mass and elemental composition ofthe fragment ions were determined. The collision induced dissociation (CID) data ofthe [M+H]~+ions provided fragmentation pathways of related compounds. Resultsshowed that the major cleavage pathways of paclitaxel and docetaxel were the same.The assay method we have developed, rapid and sensitive, was suitable for thedetermination of the low concentrations of paclitaxel in biological samples. By meansof MS scan, we could know that paclitaxel and docetaxel had the similar clearagemodes. The structural information we got were applicable to the structural elucidationand quantitative analysis of taxol.