title: Development and validation of an assay for the quantitative determination of cladribine nucleotides in MDCKII cells and culture medium using weak anion-exchange liquid chromatography coupled with tandem mass spectrometry.
abstract: The development and validation of an assay for the quantitative analysis of cladribine mono-, di- and triphosphate (2-chloro, 2'-deoxyadenosine 5'-mono-, di- and triphosphate or 2CdAMP, 2CdADP and 2CdATP) in culture medium (Optimem) and cell lysate is described. Cladribine mono- and diphosphate reference compounds were obtained by thermal degradation of cladribine triphosphate. The reference compounds were characterized using ion-pairing reversed-phase high-performance liquid chromatography with ultraviolet detection. The bioanalytical assay for 2CdAMP, 2CdADP and 2CdATP is based on weak anion-exchange liquid chromatography coupled with tandem mass spectrometry in the positive ion mode (WAXLC/MS/MS). A fused-silica electrospray capillary was used instead of a stainless steel electrospray capillary to minimize adsorption of analytes and thus decrease variation in the analyte signals. Dynamic ranges of 1.11-27.7, 0.550-55.0 and 1.31-52.3 nM for 2CdAMP, 2CdADP and 2CdATP, respectively, were validated in culture medium and cell lysate. Optimem samples required stabilization with 30% methanol to prevent conversion of 2CdATP into 2CdAMP and 2CdADP. All intra- and interday accuracies and precisions were within +/-20%. The stability of the compounds was assessed under various analytically relevant conditions. The method was successfully used to investigate cladribine nucleotide transport in vitro in Madin-Darby canine kidney II (MDCKII) cells.
authors.affiliation: Department of Pharmacy & Pharmacology, Slotervaart Hospital/The Netherlands Cancer Institute, Louwesweg 6, Amsterdam, The Netherlands. robert.jansen@slz.nl
authors.forename: Robert S
authors.initials: RS
authors.lastname: Jansen
authors.forename: Hilde
authors.initials: H
authors.lastname: Rosing
authors.forename: Cocky J F
authors.initials: CJ
authors.lastname: de Wolf
authors.forename: Jos H
authors.initials: JH
authors.lastname: Beijnen
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000494:pharmacology; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D017338:Cladribine / Q000737:chemistry* / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000737:chemistry*; D004285:Dogs; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D009711:Nucleotides / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000970:Antineoplastic Agents; D017077:Culture Media, Conditioned; D009711:Nucleotides; D017338:Cladribine