title: Use of designed experiments for the improvement of pre-analytical workflow for the quantification of intracellular nucleotides in cultured cell lines.
abstract: The present study is focused on the development of a pre-analytical strategy for the quantification of intracellular nucleotides from cultured cell lines. Different protocols, including cell recovery, nucleotide extraction and purification, were compared on a panel of nucleoside mono-, di- and triphosphates from four cell lines (adherent and suspension cells). The quantification of nucleotides was performed using a validated technique with on-line solid-phase extraction coupled with liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS/MS). Designed experiments were implemented to investigate, in a rigorous and limited-testing experimental approach, the influence of several operating parameters. Results showed that the technique used to harvest adherent cells drastically affected the amounts of intracellular nucleotides. Scraping cells was deleterious because of a major leakage (more than 70%) of intracellular nucleotides during scraping. Moreover, some other tested conditions should be avoided, such as using pure methanol as extraction solvent (decrease over 50% of intracellular nucleotides extracted from NCI-H292 cells) or adding a purification step with chloroform. Designed experiments allowed identifying an interaction between the percentage of methanol and the presence of chloroform. The mixture methanol/water (70/30, v/v) was considered as the best compromise according to the nucleoside mono-, di-, or triphosphates and the four cell lines studied. This work highlights the importance of pre-analytical step combined with the cell lines studied associated to sensitive and validated assay for the quantification of nucleotides in biological matrices.
authors.affiliation: Hospices Civils de Lyon, Centre Hospitalier Lyon-Sud, Laboratoire de Biochimie et Toxicologie, F-69495 Pierre-Bénite, France; Université de Lyon, Université Lyon 1, ISPB Faculté de pharmacie, Laboratoire de Chimie Analytique, F-69008 Lyon, France.
authors.forename: Christelle
authors.initials: C
authors.lastname: Machon
authors.affiliation: Université de Lyon, Université Lyon 1, Institut des Sciences Analytiques, UMR 5280 CNRS, Villeurbanne, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Bordes
authors.affiliation: INSERM U1052 - CNRS 5286, Centre de Recherche en Cancérologie de Lyon, F-69000 Lyon, France.
authors.forename: Emeline
authors.initials: E
authors.lastname: Cros-Perrial
authors.affiliation: Université de Lyon, Université Lyon 1, Institut des Sciences Analytiques, UMR 5280 CNRS, Villeurbanne, France.
authors.forename: Yohann
authors.initials: Y
authors.lastname: Clement
authors.affiliation: Université de Lyon, Université Lyon 1, ISPB Faculté de pharmacie, Laboratoire de Chimie Analytique, F-69008 Lyon, France; INSERM U1052 - CNRS 5286, Centre de Recherche en Cancérologie de Lyon, F-69000 Lyon, France.
authors.forename: Lars Petter
authors.initials: LP
authors.lastname: Jordheim
authors.affiliation: Université de Lyon, Université Lyon 1, Institut des Sciences Analytiques, UMR 5280 CNRS, Villeurbanne, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Lanteri
authors.affiliation: Hospices Civils de Lyon, Centre Hospitalier Lyon-Sud, Laboratoire de Biochimie et Toxicologie, F-69495 Pierre-Bénite, France; Université de Lyon, Université Lyon 1, ISPB Faculté de pharmacie, Laboratoire de Toxicologie, F-69008 Lyon, France. Electronic address: jerome.guitton@univ-lyon1.fr.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Guitton
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002725:Chloroform; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D000432:Methanol; D051379:Mice; D009711:Nucleotides / Q000032:analysis*; D052616:Solid Phase Extraction; D012997:Solvents; D053719:Tandem Mass Spectrometry / Q000379:methods; D014867:Water / Q000737:chemistry
chemical_list: D009711:Nucleotides; D012997:Solvents; D014867:Water; D002725:Chloroform; D000432:Methanol
keywords: Designed experiments; Extraction; Intracellular nucleotides; Pre-analytical workflow