title: Assay for determination of daunorubicin in cancer cells with multidrug resistance phenotype.
abstract: A sensitive assay for direct determination of intracellular level of daunorubicin (DRN) in resistant leukemia cells with overexpressed P-glycoprotein has been developed. This assay is based on a rapid separation of cells from media and fast cut-off of DRN transportation by centrifugation of cells through a layer of silicone oil. Cell pellets were extracted using 1% (v/v) formic acid in 50% (v/v) ethanol in water. The cell extracts were subsequently analysed by liquid chromatography (HPLC) coupled a low-energy collision tandem mass spectrometer equipped with an electrospray ionization source (ESI-CID-MS/MS) operated in the multiple-reaction monitoring (MRM) mode. Calibration curve was linear from 0.4 to 250nM with correlation coefficient (r²) better than 0.998. The limit of quantitation (LOQ) was 0.4 nM. The assay has been successfully applied to a determination of intracellular content of daunorubicin in sensitive K562 and resistant K562/Dox and K562/HHT300 cells.
authors.affiliation: Department of Biology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc 77515, Czech Republic.
authors.forename: Petra
authors.initials: P
authors.lastname: Krumpochova
authors.forename: Andrea
authors.initials: A
authors.lastname: Kocurova
authors.forename: Petr
authors.initials: P
authors.lastname: Dolezel
authors.forename: Petr
authors.initials: P
authors.lastname: Mlejnek
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000032:analysis / Q000493:pharmacokinetics; D004273:DNA, Neoplasm / Q000032:analysis; D003630:Daunorubicin / Q000032:analysis* / Q000493:pharmacokinetics; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D005434:Flow Cytometry; D005453:Fluorescence; D006801:Humans; D042541:Intracellular Space / Q000737:chemistry / Q000378:metabolism; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy* / Q000378:metabolism*; D016014:Linear Models; D012680:Sensitivity and Specificity
chemical_list: D000903:Antibiotics, Antineoplastic; D004273:DNA, Neoplasm; D003630:Daunorubicin