Simultaneous analysis of eight nucleoside triphosphates in cell lines by liquid chromatography coupled with tandem mass spectrometry

Cohen, Sabine; Megherbi, Mehdi; Jordheim, Lars Petter; Lefèbvre, Isabelle; Philouze, Christian; Dumontet, Charles; Guitton, Jérôme

doi:10.1016/j.jchromb.2009.09.030

Cited by 68 publications

(80 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…All values were much higher than the solid-phase extraction recoveries of NTP and dNTP reported earlier [3]. After a simple protein precipitation, recoveries ranged from 60 to 100 % [12,13,34]. …”

Section: Online Extraction Recoverymentioning

confidence: 57%

“…The first step of the analytical workflow for the study of intracellular nucleotides consists in a protein precipitation using various solvents such as 60 or 70 % methanol, trichloroacetic acid or perchloric acid (review in [13]). Thereafter, some authors perform a solid-phase extraction using anion exchange cartridge to isolate selectively nucleotides or phosphate metabolites of nucleoside analogs [3,[14][15][16].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Fully validated assay for the quantification of endogenous nucleoside mono- and triphosphates using online extraction coupled with liquid chromatography–tandem mass spectrometry

et al. 2014

Self Cite

View full text Add to dashboard Cite

An analytical method coupling online solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed to quantify 16 endogenous nucleoside mono- and triphosphates in cellular samples. Separation was achieved on a porous graphitic carbon (PGC) column without ion-pairing agent in the mobile phase. Low levels of the ion-pairing agent diethylamine (DEA) added to the reconstitution solution were necessary to prevent peak tailing of nucleoside triphosphates. The mass spectrometer, a triple quadrupole with an electrospray ionisation source, was operated in positive mode. Two multiple reaction monitoring (MRM) segments were programmed, each an internal standard. Extraction and separation of nucleoside mono- and triphosphates were obtained within 20 min. The total duration of a single run was 37 min. Calibration curves, performed with labelled nucleotides added to the sample matrix, ranged from 0.29 to 18.8 pmol injected for deoxyribonucleotides and from 3.9 to 3,156 pmol for ribonucleotides. Accuracy did not deviate more than -14.6 and 10.2 % from nominal values for all compounds at all levels. CV results were all lower than 17.0 % for the LLOQ level and 14.6 % for the other levels. Quality control (QC) samples were also in agreement with acceptance criteria, except for the lower QC of GMP. Ion suppression, matrix effect, extraction recoveries and stability were assessed. After validation, the method was applied to the evaluation of the effects of gemcitabine and hydroxyurea on nucleotide pools in Messa cells.

show abstract

Section: Online Extraction Recoverymentioning

confidence: 57%

Section: Introductionmentioning

confidence: 99%

Fully validated assay for the quantification of endogenous nucleoside mono- and triphosphates using online extraction coupled with liquid chromatography–tandem mass spectrometry

et al. 2014

Self Cite

View full text Add to dashboard Cite

show abstract

“…A highly accurate gradient LC-MS method developed for dNTP/NTPs takes 50 min using a PGC column (total cycle time 68 min), but required a mass spectrometer (MS) to distinguish all the compounds [8]. Several published methods are unable to separate ATP and dGTP [7,34,35], and in a previous paper, the very large ATP co-eluted with dTTP in cell samples [36].…”

Section: Comparison With Other Methodsmentioning

confidence: 99%

“…Validated LC-MS methods have been presented, e.g. using ion pair interactions [7] or alternative stationary phases such as porous graphite [8]. However, these methods cannot separate all dNTP/NTPs with chromatography alone.…”

Section: Introductionmentioning

confidence: 99%

Hydrophilic interaction chromatography of nucleoside triphosphates with temperature as a separation parameter

Johnsen

Wilson

Odsbu

et al. 2011

Journal of Chromatography A

View full text Add to dashboard Cite

Eight deoxynucleoside triphosphates (dNTPs) and nucleoside triphosphates (NTPs): ATP, CTP, GTP, UTP, dATP, dCTP, dGTP and dTTP, were separated with two 15 cm ZIC-pHILIC columns coupled in series, using LC-UV instrumentation. The polymer-based ZIC-pHILIC column gave significantly better separations and peak shape than a silica-based ZIC-HILIC column. Better separations were obtained with isocratic elution as compared to gradient elution. The temperature markedly affected the selectivity and could be used to fine tune separation. The analysis time was also affected by temperature, as lower temperatures surprisingly reduced the retention of the nucleotides. dNTP/NTP standards could be separated in 35 min with a flow rate of 200 μL/min. In Escherichia coli cell culture samples dNTP/NTPs could be selectively separated in 7 0min using a flow rate of 100 μL/min.

show abstract

“…In addition, nucleoside triphosphates are strongly hydrophilic compounds with similar structures which cannot be sufficiently separated under routine RP-HPLC conditions. Therefore, in recent decades, different approaches were applied to determine NTPs and/or dNTPs, including ionpair chromatography [16][17][18], capillary electrophoresis [19], hydrophilic interaction chromatography with diodearray mass spectrometric detection [20,21]. Although gas chromatography has proved to be a powerful tool for determination of compounds with low molecular weight in cell extracts [22], it has not been applied to this research because of the low volatility of the analytes.…”

Section: Introductionmentioning

confidence: 99%

An Optimized Ion-Pair HPLC Method for Simultaneous Analysis of Nucleoside Triphosphate Levels in Hepatoma Cell Line

et al. 2011

View full text Add to dashboard Cite

An improved ion-pair HPLC method was developed for the simultaneous determination of ribonucleoside triphosphates and their corresponding deoxyribonucleoside triphosphates in HepG2 cell extracts. HPLC conditions, flow rate and column temperature were optimized and good linearity (r 2 [ 0.9993) was obtained over the investigated concentration ranges. Reproducibility was evaluated by intra-and inter-day assays and RSD values were below 5.39%. Recoveries ranged from 98.2 ± 3.49% to 103.1 ± 1.75%, respectively. Finally, the method was successfully applied to the analysis of eight compounds present in HepG2 cell extracts.

show abstract

Simultaneous analysis of eight nucleoside triphosphates in cell lines by liquid chromatography coupled with tandem mass spectrometry

Cited by 68 publications

References 44 publications

Fully validated assay for the quantification of endogenous nucleoside mono- and triphosphates using online extraction coupled with liquid chromatography–tandem mass spectrometry

Fully validated assay for the quantification of endogenous nucleoside mono- and triphosphates using online extraction coupled with liquid chromatography–tandem mass spectrometry

Hydrophilic interaction chromatography of nucleoside triphosphates with temperature as a separation parameter

An Optimized Ion-Pair HPLC Method for Simultaneous Analysis of Nucleoside Triphosphate Levels in Hepatoma Cell Line

Contact Info

Product

Resources

About