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J Chromatogr A. 2015 Jul 31;1405:116-25. doi: 10.1016/j.chroma.2015.05.069. Epub 2015 Jun 6.

Use of designed experiments for the improvement of pre-analytical workflow for the quantification of intracellular nucleotides in cultured cell lines.

Author information

1
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.
2
Université de Lyon, Université Lyon 1, Institut des Sciences Analytiques, UMR 5280 CNRS, Villeurbanne, France.
3
INSERM U1052 - CNRS 5286, Centre de Recherche en Cancérologie de Lyon, F-69000 Lyon, France.
4
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.
5
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.

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.

KEYWORDS:

Designed experiments; Extraction; Intracellular nucleotides; Pre-analytical workflow

PMID:
26094139
DOI:
10.1016/j.chroma.2015.05.069
[Indexed for MEDLINE]

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