Catherine Feliu

Hélène Peyret

Damien Vautier

Zoubir Djerada
email: zoubir.djerada@univ-reims.fr

Dr Zoubir Djerada Pharmd

Simultaneous quantification of 8 nucleotides and adenosine in cells and their medium using UHPLC-HRMS

Keywords: nucleotides, adenosine, quantification, high-resolution mass spectrometry, extracellular, intracellular

Purinergic signalling is involved in physiological processes, particularly during ischemiareperfusion injuries for which it has a protective effect. The purpose of this work was to develop a method for simultaneous quantification of eight nucleotides and adenosine in biological matrices by liquid chromatography coupled with high-resolution mass spectrometry. A method was developed that was sufficiently robust to quantify the targeted analytes in 20 minutes with good sensitivity. Analysis of extracellular media from cultured endothelial cells detected the release of nucleotides and adenosine during 2 hours of hypoxia.

The quantification of cylic adenosine monophosphate (cAMP) allowed to establish a doseresponse curve after receptor stimulation. Therefore, our method allows us to study the involvement of nucleotides in various processes in both the intracellular and extracellular compartment.

Introduction

Nucleotides such as adenosine triphosphate (ATP) and adenosine are involved in vital cellular functions, including DNA and RNA synthesis, and are co-substrates in enzymatic reactions and energy production. In addition to their intracellular role, nucleotides, and adenosine have an essential role as extracellular signalling molecules known as purinergic signalling.

Extracellular nucleotides are mostly metabolised by ectonucleotidases expressed at the cell surface [START_REF] Yegutkin | Nucleotide-and nucleoside-converting ectoenzymes: Important modulators of purinergic signalling cascade[END_REF]. ATP is metabolised to adenosine diphosphate (ADP), adenosine monophosphate (AMP) and then to adenosine [START_REF] Rudolph | The biochemistry and physiology of nucleotides[END_REF]. Extracellular nucleotides and extracellular adenosine are involved in physiological processes via the P2 and P1 receptors, respectively [START_REF] Abbracchio | Purinergic signalling: pathophysiological roles[END_REF][START_REF] Burnstock | Purinergic Signaling in the Cardiovascular System[END_REF]. Our laboratory is interested in the processes of ischemia-reperfusion of heart and vessels. It is well known that extracellular nucleotides and nucleosides can provide a protective effect against ischemia-reperfusion injuries [START_REF] Djerada | Current knowledge on the role of P2Y receptors in cardioprotection against ischemia-reperfusion[END_REF][START_REF] Djerada | Extracellular NAADP affords cardioprotection against ischemia and reperfusion injury and involves the P2Y11-like receptor[END_REF][START_REF] Cohen | Adenosine: trigger and mediator of cardioprotection[END_REF][START_REF] Feliu | Ticagrelor Prevents Endothelial Cell Apoptosis through the Adenosine Signalling Pathway in the Early Stages of Hypoxia[END_REF][START_REF] Feliu | Complementary Role of P2 and Adenosine Receptors in ATP Induced-Anti-Apoptotic Effects Against Hypoxic Injury of HUVECs[END_REF]. Intracellular concentrations of ATP range from 5 to 8 mM while extracellular concentrations are in the nanomolar or micromolar range under physiological conditions [START_REF] Yegutkin | Nucleotide-and nucleoside-converting ectoenzymes: Important modulators of purinergic signalling cascade[END_REF][START_REF] Gorman | Human Plasma ATP Concentration[END_REF]. Nucleotides released into extracellular medium exert their activity by binding to purinergic receptors expressed on the surface of different cell types.

The hydrolysis of nucleotides by ectonucleotidases plays an important role in determining extracellular concentrations of nucleotides and nucleosides and consequently in their actions on receptors.

Purinergic signalling is well established in cytoprotection, particularly in cardioprotection [START_REF] Burnstock | Purinergic Signaling in the Cardiovascular System[END_REF][START_REF] Djerada | Current knowledge on the role of P2Y receptors in cardioprotection against ischemia-reperfusion[END_REF], renal protection [START_REF] Burnstock | Purinergic signalling in the kidney in health and disease[END_REF], cerebral protection [START_REF] Pedata | Purinergic signalling in brain ischemia[END_REF] and endothelial protection [START_REF] Burnstock | Purinergic signaling and blood vessels in health and disease[END_REF][START_REF] Ralevic | Purinergic transmission in blood vessels[END_REF].

The quantitative analysis of intracellular, extracellular and tissue nucleotides is therefore of major relevance for the study of purinergic signalling and cytoprotection. For the quantification of ATP, described assay are based on bioluminescence, colorimetric or fluorescent techniques [START_REF] Gorman | Human Plasma ATP Concentration[END_REF][START_REF] Gorman | Measurement of adenine nucleotides in plasma[END_REF]. However, due to the physicochemical complexity of the samples and a large number of compounds candidates to quantify, a separation method such as high-performance liquid chromatography is required. The detection modes used are the ultraviolet detector [START_REF] Contreras-Sanz | Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC[END_REF][START_REF] Cichna | Determination of fifteen nucleotides in cultured human mononuclear blood and umbilical vein endothelial cells by solvent generated ion-pair chromatography[END_REF], the diode array detector [START_REF] Fukuuchi | Analysis of Intra-and Extracellular Levels of Purine Bases, Nucleosides, and Nucleotides in HepG2 Cells by High-performance Liquid Chromatography[END_REF] and the mass spectrometric detector of which is the most sensitive and specific [START_REF] Machon | Fully validated assay for the quantification of endogenous nucleoside mono-and triphosphates using online extraction coupled with liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Klawitter | Development and validation of an assay for the quantification of 11 nucleotides using LC/LC-electrospray ionization-MS[END_REF][START_REF] Cordell | Quantitative profiling of nucleotides and related phosphate-containing metabolites in cultured mammalian cells by liquid chromatography tandem electrospray mass spectrometry[END_REF][START_REF] Chen | A LC-MS/MS method for the analysis of intracellular nucleoside triphosphate levels[END_REF][START_REF] Cohen | Simultaneous analysis of eight nucleoside triphosphates in cell lines by liquid chromatography coupled with tandem mass spectrometry[END_REF][START_REF] Jiang | Simultaneous determination of adenine nucleotides, creatine phosphate and creatine in rat liver by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry[END_REF][START_REF] Pabst | Nucleotide and nucleotide sugar analysis by liquid chromatography-electrospray ionization-mass spectrometry on surface-conditioned porous graphitic carbon[END_REF][START_REF] Peifer | Quantitation of intracellular purine intermediates in different Corynebacteria using electrospray LC-MS/MS[END_REF][START_REF] Thomas | Quantitation of endogenous nucleoside triphosphates and nucleosides in human cells by liquid chromatography tandem mass spectrometry[END_REF][START_REF] Wang | Simultaneous determination of creatine phosphate, creatine and 12 nucleotides in rat heart by LC-MS/MS[END_REF][START_REF] Zhu | A simultaneously quantitative method to profiling twenty endogenous nucleosides and nucleotides in cancer cells using UHPLC-MS/MS[END_REF][START_REF] Cortese | Validation of a LC/MSMS method for simultaneous quantification of 9 nucleotides in biological matrices[END_REF][START_REF] He | Simultaneous Quantification of Nucleosides and Nucleotides from Biological Samples[END_REF][START_REF] Zhang | Strategies for quantitation of endogenous adenine nucleotides in human plasma using novel ion-pair hydrophilic interaction chromatography coupled with tandem mass spectrometry[END_REF].

Due to significant differences in polarity between compounds, the simultaneous quantification of analytes such as nucleosides (e.g. adenosine), monophosphate nucleotides (AMP, AMPc and UMP), biphosphate (ADP and UDP) and triphosphate (ATP and UTP) is very challenging. Ion-pairing agents such as tetrabutylammonium hydroxide and hexylamine have been widely used for the quantification of nucleosides and nucleotides to achieve satisfactory retention and peak shape particularly for nucleotides [START_REF] Klawitter | Development and validation of an assay for the quantification of 11 nucleotides using LC/LC-electrospray ionization-MS[END_REF][START_REF] Cordell | Quantitative profiling of nucleotides and related phosphate-containing metabolites in cultured mammalian cells by liquid chromatography tandem electrospray mass spectrometry[END_REF][START_REF] Chen | A LC-MS/MS method for the analysis of intracellular nucleoside triphosphate levels[END_REF][START_REF] Cohen | Simultaneous analysis of eight nucleoside triphosphates in cell lines by liquid chromatography coupled with tandem mass spectrometry[END_REF][START_REF] Wang | Simultaneous determination of creatine phosphate, creatine and 12 nucleotides in rat heart by LC-MS/MS[END_REF][START_REF] Zhu | A simultaneously quantitative method to profiling twenty endogenous nucleosides and nucleotides in cancer cells using UHPLC-MS/MS[END_REF][START_REF] Zhang | Strategies for quantitation of endogenous adenine nucleotides in human plasma using novel ion-pair hydrophilic interaction chromatography coupled with tandem mass spectrometry[END_REF]. However, these agents produced an ion-suppressive effect and reduced sensitivity. In addition, cleaning of the mass spectrometer was required more frequently. Recently, some teams have proposed their work without using these ion-pairing agents [START_REF] Machon | Fully validated assay for the quantification of endogenous nucleoside mono-and triphosphates using online extraction coupled with liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Jiang | Simultaneous determination of adenine nucleotides, creatine phosphate and creatine in rat liver by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry[END_REF][START_REF] Pabst | Nucleotide and nucleotide sugar analysis by liquid chromatography-electrospray ionization-mass spectrometry on surface-conditioned porous graphitic carbon[END_REF][START_REF] Thomas | Quantitation of endogenous nucleoside triphosphates and nucleosides in human cells by liquid chromatography tandem mass spectrometry[END_REF][START_REF] Cortese | Validation of a LC/MSMS method for simultaneous quantification of 9 nucleotides in biological matrices[END_REF][START_REF] He | Simultaneous Quantification of Nucleosides and Nucleotides from Biological Samples[END_REF]. In this field, a porous graphitic carbon (PGC) column allowing good retention of highly polar compounds have emerged [START_REF] Machon | Fully validated assay for the quantification of endogenous nucleoside mono-and triphosphates using online extraction coupled with liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Jiang | Simultaneous determination of adenine nucleotides, creatine phosphate and creatine in rat liver by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry[END_REF][START_REF] Pabst | Nucleotide and nucleotide sugar analysis by liquid chromatography-electrospray ionization-mass spectrometry on surface-conditioned porous graphitic carbon[END_REF]. Among all these works, only four have proposed the simultaneous quantification of nucleotides and nucleosides [START_REF] Cordell | Quantitative profiling of nucleotides and related phosphate-containing metabolites in cultured mammalian cells by liquid chromatography tandem electrospray mass spectrometry[END_REF][START_REF] Thomas | Quantitation of endogenous nucleoside triphosphates and nucleosides in human cells by liquid chromatography tandem mass spectrometry[END_REF][START_REF] Zhu | A simultaneously quantitative method to profiling twenty endogenous nucleosides and nucleotides in cancer cells using UHPLC-MS/MS[END_REF][START_REF] He | Simultaneous Quantification of Nucleosides and Nucleotides from Biological Samples[END_REF]. These publications proposed the quantification of nucleosides and nucleotides on cells [START_REF] Cordell | Quantitative profiling of nucleotides and related phosphate-containing metabolites in cultured mammalian cells by liquid chromatography tandem electrospray mass spectrometry[END_REF][START_REF] Thomas | Quantitation of endogenous nucleoside triphosphates and nucleosides in human cells by liquid chromatography tandem mass spectrometry[END_REF][START_REF] Zhu | A simultaneously quantitative method to profiling twenty endogenous nucleosides and nucleotides in cancer cells using UHPLC-MS/MS[END_REF] or in plasma and urine [START_REF] He | Simultaneous Quantification of Nucleosides and Nucleotides from Biological Samples[END_REF].

Analytical difficulties also increase when the assay is performed in a medium containing high-polarity components, such as cell culture medium or ex-vivo exploration fluids (e. g.

Krebs solution in the isolated infused heart model, study of isolated arteries in arteriography)

and solutions used in microdialysis techniques. Indeed, these media are rich in salt and glucose, which leads to competition at the interaction sites of the stationary phase inducing inappropriate elution profiles and less ionisation at the source of the mass spectrometer thus limiting the sensitivity of the procedure. To our knowledge, to date, no author has described a method that can quantify in these environments at nanomolar (nM) concentrations. This method should also apply to quantification in the environments usually described, such as cells, tissues, and organs.

The objective of this study was to develop and validate a method for quantifying eight nucleotides (ATP, ADP, AMP, cAMP, cADP-ribose, UTP, UDP and UMP) and adenosine.

The method has been optimised to allow the quantification of compounds in the same extraction and chromatographic analysis. Using a PGC column (porous graphitic carbon) and a mobile phase with an extreme basic pH, this optimised process was applied to three different matrices, supernatants from human cultured cells, cardiomyocytes and rat tissues to measure endogenous nucleoside and nucleotide concentrations.

Materials and methods

Chemical and reagents

Adenosine, adenosine 5′-monophosphate (AMP), cyclic adenosine monophosphate (cAMP), adenosine 5′-diphosphate (ADP), cyclic adenosine 5′-diphosphate ribose (cADP-ribose), adenosine 5′-triphosphate (ATP), uridine 5′-monophosphate (UMP), uridine 5′-diphosphate (UDP), uridine 5′-triphosphate (UTP), nicotinamide D4 (internal standard), ammonium acetate and ascorbic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Acetonitrile, methanol and water, all hypergrade LC-MS for mobile phase, were purchased from Biosolve (Dieuze, France). Ammonium hydroxide was purchased from Merck (Darmstadt, Germany).

Stock solutions of compounds of interest and internal standards were prepared by dissolving the compounds in water to a final concentration of 1 g.L -1 and stored for one month at -20 °C.

The working solutions were diluted with an appropriate volume of water to obtain different concentration levels ranging from 0.039 to 5 mg.L -1 . The concentration of the working solution of the internal standard was 0.1 mg.L -1 . The calibration standard (STD) was prepared by adding 10 µL of working solutions and 10 µL of nicotinamide D4 to the matrix (Krebs solution). Finally, the calibration ranged from 7.8 to 1000 µg.L -1 corresponding to 1.

Instrumentation

A high-performance liquid chromatography system with an Ultimate 3000 high-pressure pump coupled with an Orbitrap QExactive mass spectrometer (ThermoFisher Scientific, San Jose, CA, USA) was used to develop and validate the method. TraceFinder Forensic 3.3 was used for LC-MS, acquisition, and processing.

Chromatographic separation was performed using an UHPLC (ThermoFisher Scientific, San Jose, CA, USA) column of Hypercarb 5 μm (2.1 × 150 mm), maintained at 35 °C. The mobile phases consisted of water + 2 mM ammonium acetate pH 10 adjusted with ammonia (mobile phase A), acetonitrile (mobile phase B) and water + ascorbic acid 50 mg.L -1 (mobile phase C).

A programmed mobile phase gradient was used at a flow rate of 0.3 mL/min (Table 2). The analysis and acquisition time was 20 minutes, including equilibration.

Detector coupling was performed using a heated electrospray ionisation source (HESI) operating in positive ionisation mode, with the following settings: sheat gas 53 arbitrary units (AU), auxiliary gas 14 AU; sweep gas flow rate 3 AU; spray voltage 3.30 kV; ion transfer capillary temperature 250 °C; S-lens RF level 70 AU and heating temperature 275 °C. Mass spectrometry was performed using the full scanning mode in positive and negative polarity and a data-dependent acquisition mode (ddMS²). The execution acquisition was 5.5 to 10.55 min. The acquisition parameters were as follows: high resolution of 70.000 at m/z 200 (resolution was calculated at full width at maximum mid-height); automatic gain control (AGC) target 1e6 AU; maximum injection time (IT) 100 ms. For the ddMS² mode, an inclusion list including interest compounds has been added. The ddMS² mode settings were as follows: the resolution 17,500 at m/z 200; automatic gain control (AGC) target 1e5 AU; maximum injection time (IT) 50 ms; isolation window 2.0 m/z. Mass calibration was performed once a week in a positive and negative mode according to the manufacturer's recommendations using an external calibration solution (Pierce®, ThermoFisher Scientific, San Jose, CA, USA).

Sample preparation

Cell culture

Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell and cultured in an endothelial cell growth medium (PromoCell, Sickingenstraße, Heidelberg, Germany) containing 2 % (vol/vol) SCC, 0.4 % (vol/vol) endothelial growth supplement: 0.1 ng/mL human EGF, 1.0 μg/mL hydrocortisone, 1 ng/mL human bFGF, 90 µg/mL heparin and 1 % (vol/vol) penicillin/streptomycin (DUTSCHER SAS, Brumath, France) in a humidified atmosphere at 37 °C and 5 % CO2. The medium was changed three times a week. Confluent cultures were detached using the PromoCell detachment kit containing HepesBSS (30 mM Hepes), Trypsin / EDTA Solution (0.04 % / 0.03 %) and Trypsin Neutralising Solution (TNS). The cells were seeded at 6,000 cells per cm 2 . The FCS was reduced to 1 % 24 hours before the experiment. All experiments were performed with subconfluent endothelial monolayers in the fourth passage.

Subconfluent monolayers of endothelial cells (80 %) were exposed to 2 hours of hypoxia. The culture medium was replaced by a Hank serum-free buffered saline solution (HBSS; PAA), and the cells were placed in a custom hypoxic chamber (Bactron, Sheldon Manufacturing Inc, Cornelius, NC, USA). The cells were exposed to 95 % N2 and 5 % CO2 for 2 hours at 37 °C.

The partial pressure of oxygen (pO2) in the hypoxic chamber was reduced to less than 2 % during the experiment. Cell culture media were collected during the experiment at 8 different times (T0 min, T15 min, T30 min, T60 min, T120 min, T135 min, T180 min, and T240 min).

Cells were also harvested after hypoxia and reoxygenation.

For rat cardiomyocyte culture, the ventricular tissues are first washed in a saline solution of Hank without calcium or magnesium (CMF HBSS) and incubated overnight with 50 ng/mL of trypsin, then with an excess of 5 M of soybean trypsin inhibitor. The tissues were then treated with 300 U/mL collagenase in HBSS CMF for 45 minutes under constant agitation.

After crushing to release the cells, the cell suspension was filtered (70 µm pores) and centrifuged for 5 minutes at 100 g. Primary neonatal cardiomyocytes were plated at 125,000 cells per cm 2 in 6-well plates and cultured for 3 days in L-15 Leibovitz medium (Worthington Biochemical Corporation, Reading, UK) with 5 % (vol/vol) fetal calf serum and 1 % (vol/vol) penicillin and streptomycin (Life Technologies, Invitrogen, Cergy-Pontoise, France)

Treatment of extracellular medium

The culture medium (50 μL) was recovered at different times during the experiment. As described, to inhibit ectonucleotidase activity 75 μL (60 % V/V) methanol was added and the extracts were frozen at -80 °C [START_REF] Cohen | Simultaneous analysis of eight nucleoside triphosphates in cell lines by liquid chromatography coupled with tandem mass spectrometry[END_REF][START_REF] Zhang | Strategies for quantitation of endogenous adenine nucleotides in human plasma using novel ion-pair hydrophilic interaction chromatography coupled with tandem mass spectrometry[END_REF][START_REF] Canelas | Quantitative evaluation of intracellular metabolite extraction techniques for yeast metabolomics[END_REF]]. An internal standard solution (nicotinamide D4 10 μg.L -1 ) was added. The mixtures were evaporated under nitrogen at 40 °C and then reconstituted with 100 μL of ice water. Twenty microlitres were injected for chromatographic separation.

Treatment of cultured neonatal rat cardiomyocyte or ventricular heart rat tissue

Extraction was performed according to Cordell et al. and Djerada et al. [21,[START_REF] Djerada | Intracellular NAADP increase induced by extracellular NAADP via the P2Y11-like receptor[END_REF]. The cells or ventricular heart tissue were washed with cold PBS. Methanol (500 µL per 6-well plate well) was added to the pellet and the cells were scraped. The heart tissue was crushed in ice. After three times ten seconds of sonication, the samples were centrifuged (4 °C, 30 min, 16 000 g).

The supernatant (50 µL) was recovered and stored immediately at -80 °C until analysis. For the quantification analysis, 10 µL of the working solution of the internal standard was added to the sample. After evaporation under nitrogen flow at 40 °C, the dry extract was reconstituted with 100 μL of ice-cold water (LC-MS hypergrade). Twenty microlitres were injected for chromatographic separation. All the animal experiment procedure was approved by the Animal Ethics Committee of Reims.

Validation procedure

The method developed for the quantification of nucleotides and adenosine has been validated in accordance with international guidelines in terms of linearity, precision, accuracy, matrix effect and stability [START_REF]Bioanalytical method validation[END_REF][START_REF] Emea | Guideline on bioanalytical method validation[END_REF] as previously described [START_REF] Djerada | Validation of a fast method for quantitative analysis of elvitegravir, raltegravir, maraviroc, etravirine, tenofovir, boceprevir and 10 other antiretroviral agents in human plasma samples with a new UPLC-MS/MS technology[END_REF][START_REF] Feliu | Validation of a fast UPLC-MS/MS method for quantitative analysis of opioids, cocaine, amphetamines (and their derivatives) in human whole blood[END_REF][START_REF] Cazaubon | Population pharmacokinetics of articaine with 1:200,000 epinephrine during third molar surgery and simulation of high-dose regimens[END_REF].

Linearity was tested by testing different weighting functions for each analyte to select the regression calibration (linear, 1/X, 1/X², 1/Y...) as described [START_REF] Cortese | Validation of a LC/MSMS method for simultaneous quantification of 9 nucleotides in biological matrices[END_REF]. The accuracy of the calibration standard was checked by analysing the distribution of deviations from the prediction of regression or residual value (Gaussian distribution verified by the Agostino Pearson test). Accuracy (%) was expressed in relative error (RE, %) as the ratio (mean-target value)/target value of the 6 repeated analysis. The acceptance criteria did not deviate by more than 15 % from the nominal concentration.

Precision and accuracy were assessed based on quality control at four concentrations: QC1, QC2, QC3 and QC4 (Table 1). Accuracy and precision within and between cycles were assessed by analysing 9 samples per level. The accuracy has been expressed as a relative standard deviation and must not exceed ± 15 %. For accuracy reasons, the average concentration should be within ± 15 % of the target concentration at each level tested.

The matrix effect was evaluated with six matrices of Krebs solution from different sources.

Matrices were spiked after extraction with internal standard and with analytes at 2 concentrations: 30 µg.L -1 (corresponding to 57.3 nM for ATP, 68.1 nM for ADP, 87.72 nM for AMP, 88.3 nM for cAMP, 53.5 nM for cADP-ribose, 108.7 nM for adenosine, 60.03 nM for UTP, 71.9 nM for UDP and 89.7 nM for UMP) and 800 µg.L -1 (corresponding to 1577.4 nM for ATP, 1872.6 nM for ADP, 2304 nM for AMP, 2430 nM for cAMP,1472.4 nM for cADP-ribose, 2993.5 nM for adenosine, 1652.4 nM for UTP, 1979.4 nM for UDP and 2467.8 nM for UMP). The matrix factor was calculated by comparing the area under the peak of the analyte of interest from the spiked matrix after extraction and the area under the peak of a pure solution of this analyte at the same concentration. The normalised matrix factor was also calculated for each analyte by dividing the matrix factor of the analyte by the matrix factor of the internal standard. The relative standard deviation of the normalised factors must be less than 20 % [START_REF] Emea | Guideline on bioanalytical method validation[END_REF][START_REF] Matuszewski | Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS[END_REF][START_REF] Chambers | Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses[END_REF][START_REF] Taylor | Matrix effects: the Achilles heel of quantitative high-performance liquid chromatography-electrospray-tandem mass spectrometry[END_REF].

Post-analytical stability was investigated by comparing the concentrations of analytes before and after the storage of the extracted samples at 15 °C in the autosampler for 24 hours.

Statistical analysis

All statistical tests were performed with GraphPad Prism 5.0 (San Diego, CA, USA). For applicability, the values are presented as an average ± SEM (standard error of the average).

Differences between groups were evaluated using one-way analysis of variance (ANOVA). If the F ratio was significant, Tukey's Multiple Comparison (post hoc) test was applied to assess significance (p < 0.05). In the experimentations of just two groups, the differences were assessed using unpaired t test. EC50 values for agonists were derived from data of pooled and normalised log concentration-effect curves fitted according to the nonlinear four-parameter logistic equation (Prism 4.00; GraphPad Software, San Diego, CA). p < 0.05 was considered to be statistically significant.

Results and discussion

Optimisation of the method

MS Optimisation

The negative and positive ionisation mode has been tested for the detection of analytes. Due to their high polarity and the acid nature of the targeted analytes, the negative mode was used for ATP (m/z: 505.98847), ADP (m/z: 426.02214), AMP (m/z: 346.05581), cAMP (m/z: 328.04524), cADP-ribose (m/z: 540.05383), UTP (m/z: 482.96125), UDP (m/z: 402.99492) and UMP (m/z: 323.02859) while the positive mode was used for adenosine (m/z: 268.10403).

Subsequently, different acquisition modes of the mass spectrometry were tested: full scan, full scan ddMS² and parallel reaction monitoring (PRM). The PRM mode did not allow us to obtain a satisfactory sensitivity for the further development of our work. Full scan mode, more sensitive than PRM mode, did not discriminate isomeric compounds, such as ATP and deoxyguanosine triphosphate (dGTP), ADP and deoxyguanosine diphosphate (dGDP), AMP and deoxyguanosine monophosphate (dGMP), adenosine and deoxyguanosine. Although our method does not quantify deoxyguanosine and its derivatives, these compounds are susceptible to be in our samples and may interfere. A complete scan followed by a datadependent MS/MS (ddMS²) was applied to obtain specific mass spectra for ATP, ADP, AMP, and adenosine.

Then, to obtain maximum signals, instrument parameters such as sheath gas auxiliary gas, sweep gas flow rate, spray voltage, ion transfer capillary temperature, S objective RF level and heater temperature were optimised. The settings of the mass spectrometer parameters have been listed in section 2.2.

Liquid chromatography optimisation

Selection of the column:

The first step in optimising the chromatographic conditions was the choice of the column. The main difficulties of chromatographic separation were, on the one hand, the similar structures of the different nucleotides and on the other hand, their polarities. Nucleotides are highly polar molecules that have several ionisable groups and therefore level of pKa. Depending on the pH, nucleotides will be positively or negatively ionised with one or more charges. In addition, due to their high polarity, nucleotides are not retained on conventional stationary phases such as C18. The main methods performed with stationary C18 columns allow the quantification of monophosphate nucleotides only [START_REF] Peifer | Quantitation of intracellular purine intermediates in different Corynebacteria using electrospray LC-MS/MS[END_REF][START_REF] Gill | Analysis of nucleosides and nucleotides in infant formula by liquid chromatography-tandem mass spectrometry[END_REF]. Wang et al. proposed the quantification of mono, di and triphosphate nucleotides with a C18 column with polar endcapping [START_REF] Wang | Simultaneous determination of creatine phosphate, creatine and 12 nucleotides in rat heart by LC-MS/MS[END_REF]. To overcome this difficulty, other chromatographic methods have been described to allow nucleotide separation: C18 column ion pairing, a porous graphitic carbon column chromatography, ion-exchange column chromatography and HILIC column chromatography.

Ion pairing chromatography is the most widely published method for quantifying nucleotides.

It consists of adding one or more ion-pair agents, such as hexylamine or diethylamine, in the mobile phase [START_REF] Contreras-Sanz | Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC[END_REF][START_REF] Cichna | Determination of fifteen nucleotides in cultured human mononuclear blood and umbilical vein endothelial cells by solvent generated ion-pair chromatography[END_REF][START_REF] Cordell | Quantitative profiling of nucleotides and related phosphate-containing metabolites in cultured mammalian cells by liquid chromatography tandem electrospray mass spectrometry[END_REF][START_REF] Zhang | Strategies for quantitation of endogenous adenine nucleotides in human plasma using novel ion-pair hydrophilic interaction chromatography coupled with tandem mass spectrometry[END_REF][START_REF] Graven | Purine metabolite and energy charge analysis of Trypanosoma brucei cells in different growth phases using an optimized ionpair RP-HPLC/UV for the quantification of adenine and guanine pools[END_REF]. This procedure improves nucleotide retention and peak chromatographic shape, especially with triphosphates in the stationary phase. Unfortunately, this process has the disadvantage of prematurely damaging the columns, causing ion removal effects and thus significantly reducing sensitivity.

Ion exchange chromatography is also described for nucleotide quantification. Hydrophilic interaction chromatography (HILIC) is a chromatographic technique combining the properties of normal phase chromatography, reverse phase chromatography and ion chromatography [START_REF] Zhang | Strategies for quantitation of endogenous adenine nucleotides in human plasma using novel ion-pair hydrophilic interaction chromatography coupled with tandem mass spectrometry[END_REF][START_REF] Vo Duy | A quantitative liquid chromatography tandem mass spectrometry method for metabolomic analysis of Plasmodium falciparum lipid related metabolites[END_REF]. Finally, another chromatographic assay uses columns with a stationary phase of carbon graphite or PGC column (porous graphitic carbon) [START_REF] Machon | Fully validated assay for the quantification of endogenous nucleoside mono-and triphosphates using online extraction coupled with liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Jiang | Simultaneous determination of adenine nucleotides, creatine phosphate and creatine in rat liver by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry[END_REF][START_REF] Pabst | Nucleotide and nucleotide sugar analysis by liquid chromatography-electrospray ionization-mass spectrometry on surface-conditioned porous graphitic carbon[END_REF][START_REF] Zhu | A simultaneously quantitative method to profiling twenty endogenous nucleosides and nucleotides in cancer cells using UHPLC-MS/MS[END_REF]. To date, only one published method allows the simultaneous quantification of nucleosides and nucleotides from biological samples, using Acquity HSS T3 (Waters, Milford, MA, USA) [START_REF] He | Simultaneous Quantification of Nucleosides and Nucleotides from Biological Samples[END_REF].

Different columns, known for their application to polar compounds, were tested, namely

Mobile phase optimisation:

The composition of the mobile phase influences the ionisation state of the analytes and their interaction with the stationary PGC phase, which is a major determinant of peak retention and shape [START_REF] De Matteis | Chromatographic retention behaviour of monosubstituted benzene derivatives on porous graphitic carbon and octadecyl-bonded silica studied using molecular modelling and quantitative structure-retention relationships[END_REF]. At this step of optimisation, a 100 µg.L -1 pure solution of ATP, ADP and AMP is used. Several mobile phases (ammonium acetate or ammonium formate) at different pH value were evaluated in order to obtain a good peak shape and better separation of target compounds without peak tailing.

First, ammonium acetate provided a better signal strength than ammonium formate for most targeted compounds such as di or triphosphates. Different concentrations of ammonium acetate were tested as previously described, 2 mM [START_REF] Jiang | Simultaneous determination of adenine nucleotides, creatine phosphate and creatine in rat liver by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry[END_REF], 5 mM [START_REF] Zhu | A simultaneously quantitative method to profiling twenty endogenous nucleosides and nucleotides in cancer cells using UHPLC-MS/MS[END_REF] and 10 mM [START_REF] Wang | Simultaneous determination of creatine phosphate, creatine and 12 nucleotides in rat heart by LC-MS/MS[END_REF]. The best chromatographic peaks and signals were obtained with 2 mM ammonium acetate.

The influence of pH on signal of ATP, ADP and AMP was evaluated at various pH values: 9, 9.5 and 10 (Figure 1A). At low pH, a loss of sensitivity and a peak tailing were observed with di and tri-nucleotides. These results were due to the adsorption and also to the entrapment of phosphorylated analytes on the stainless steel surface of the system [START_REF] Tuytten | Stainless steel electrospray probe: a dead end for phosphorylated organic compounds?[END_REF][START_REF] Asakawa | Suppression effects of carbonate on the interaction between stainless steel and phosphate groups of phosphate compounds in high-performance liquid chromatography and electrospray ionization mass spectrometry[END_REF]. This was observed when these compounds were introduced under acidic conditions (0.1 % acetic acid).

By applying an extreme basic mobile phase (pH 12), compounds were less entrapped [START_REF] Tuytten | Stainless steel electrospray probe: a dead end for phosphorylated organic compounds?[END_REF].

From these data, some authors have proposed procedures with basic mobile phases [START_REF] Jiang | Simultaneous determination of adenine nucleotides, creatine phosphate and creatine in rat liver by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry[END_REF][START_REF] Wang | Simultaneous determination of creatine phosphate, creatine and 12 nucleotides in rat heart by LC-MS/MS[END_REF][START_REF] Zhu | A simultaneously quantitative method to profiling twenty endogenous nucleosides and nucleotides in cancer cells using UHPLC-MS/MS[END_REF][START_REF] Asakawa | Suppression effects of carbonate on the interaction between stainless steel and phosphate groups of phosphate compounds in high-performance liquid chromatography and electrospray ionization mass spectrometry[END_REF]. As shown in Figure 1A, the increase in pH in the mobile phase improved the separation, peak shapes and signal intensity.

Under isocratic conditions (ammonium acetate 2 mM pH 10 (A)/ acetonitrile (B) at 90/10 ratio), only ATP, ADP, AMP, UTP, UDP, and UMP were retained and eluted from the column. Other ratios of the mobile phase were tested but did not provide good retention and separation of compounds with a correct chromatographic peak shape. Adenosine was eluted, but the other analytes peaks were spread out. Under isocratic conditions ammonium acetate 2 mM pH 10 / acetonitrile (50/50), the ATP chromatographic peak was tailing. With a ratio of 45/55, the ATP chromatographic peak shows an unsatisfactory shape with peak splitting. In addition to the unsatisfactory results obtained for ATP, a loss of signal and too large peaks have been observed for each compound of interest. Under isocratic conditions ammonium acetate 2 mM pH 10 / acetonitrile (60/40), the shape of the chromatographic peaks seemed satisfactory. However, the retention and separation of the compounds were not satisfactory (same retention times for ATP, ADP, and AMP at 1.30 min and adenosine retention time at 3.33 min). This step still required an optimisation phase, in particular by using an elution gradient. To obtain sharper peaks and improve separation and retention, a gradient elution program was tested. For the first step of the gradient program, the adjustment of water and ammonium acetate 2 mM to pH 10 maintained the negative charge on the phosphate groups and allowed higher interaction of the targeted compounds to the PCG column. The objective of the second gradient step was the elution of the compounds and, for this purpose, acetonitrile was used. The first gradient tested was that of the study performed by Jiang et al. [START_REF] Chen | A LC-MS/MS method for the analysis of intracellular nucleoside triphosphate levels[END_REF] (0 min, 2% B; 2 min, 2 % B; 4 min, 15 % B; 10 min, 15 % B, 13 min, 50 %). This gradient allowed good retention of nucleotides (retention times: ATP 9.15 min, ADP 9.17 min, and AMP 9.90 min) but allowed neither satisfactory separation of ATP and ADP nor elution of adenosine. In our second test, the step from 4 to 10 min at 85/15 was removed (flow rate of 0.2 mL/min: 0 min, 2% B; 2 min; 10 min, 50 %, 13 min, 50 %). This increased the slope of the gradient. Adenosine was thus eluted with this gradient (15.32 min) but ATP, with a retention time at 9.79 min showed a wide double chromatographic peak, with an unsatisfactory shape. The gradient program has been optimised. New elution gradient tests were conducted, increasing the percentage of acetonitrile in the mobile phase (0 min, 2% B; 2 min, 2 % B; 7 min, 50 % B; 10 min 50 % B, then 0 min, 2% B; 2 min, 2 % B; 7 min, 60 % B; 10 min 60 % B, and finally 0 min, 2% B; 2 min, 2 % B; 7 min, 98 % B; 10 min 98 % B).

These gradient tests allowed us to obtain a correct chromatographic peak, not large and not trailing particularly for ATP with satisfactory retention and acceptable separation (ATP 6.23 min, ADP 6.21 min, AMP 6.42 min, and adenosine 8.13 min). At this stage of the optimisation, we found a serious limitation with the PCG column consisting of a retention time delay of ATP associated with a significant loss of resolution and signal during repeated injections. As shown in Figure 1B, following repeated injections, a retention time delay of ATP associated with a significant loss of signal intensity at the third injection was observed.

After 4 injections, the signal had disappeared. This loss of retention and signal is mainly observed for triphosphorylated nucleotides. The same observations were also noted, but with lower extent, for other di-and mono-phosphorylated compounds. Bapiro et al. have reported variability and loss of retention over repeated injection cycle and over a period of time, thus limiting its use [START_REF] Bapiro | Understanding the Complexity of Porous Graphitic Carbon (PGC) Chromatography: Modulation of Mobile-Stationary Phase Interactions Overcomes Loss of Retention and Reduces Variability[END_REF]. As the injection progresses, the surface of the stationary phase can be modified, changing the retention properties, causing a loss of retention and ultimately a loss of signal intensity. The loss of retention on the PGC column is mainly related to high contamination of the column and reduction oxydation reactions [START_REF] Bapiro | Understanding the Complexity of Porous Graphitic Carbon (PGC) Chromatography: Modulation of Mobile-Stationary Phase Interactions Overcomes Loss of Retention and Reduces Variability[END_REF]. Several studies regularly propose washing procedures using strong acids/bases [START_REF] Pabst | Nucleotide and nucleotide sugar analysis by liquid chromatography-electrospray ionization-mass spectrometry on surface-conditioned porous graphitic carbon[END_REF][START_REF] Reepmeyer | Separation and detection of the isomeric equine conjugated estrogens, equilin sulfate and Δ8,9-dehydroestrone sulfate, by liquid chromatography-electrospray-mass spectrometry using carbon-coated zirconia and porous graphitic carbon stationary phases[END_REF]. The use of buffered hydrogen peroxide for column preconditioning has also been reported to prevent loss of retention [START_REF] Reepmeyer | Separation and detection of the isomeric equine conjugated estrogens, equilin sulfate and Δ8,9-dehydroestrone sulfate, by liquid chromatography-electrospray-mass spectrometry using carbon-coated zirconia and porous graphitic carbon stationary phases[END_REF][START_REF] Jansen | Retention studies of 2′-2′difluorodeoxycytidine and 2′-2′-difluorodeoxyuridine nucleosides and nucleotides on porous graphitic carbon: Development of a liquid chromatography-tandem mass spectrometry method[END_REF]. All these procedures have increased the analysis time and involve the use of hazardous chemicals without improvement of original retention. An alternative method, with a 95 % step of methanol (in water) added at the end of the gradient for 2 minutes, has been proposed by Bapiro et al. to ensure the maintenance of the initial retention capacity [START_REF] Bapiro | Understanding the Complexity of Porous Graphitic Carbon (PGC) Chromatography: Modulation of Mobile-Stationary Phase Interactions Overcomes Loss of Retention and Reduces Variability[END_REF]. This assay was tested for our application but was ineffective for retention of ATP. Figure 1C depicted the wash procedures tested at the end of the gradient to maintain ATP retention and signal. Only the use of 50 mg.L -1 ascorbic acid for 7 minutes at the end of the gradient allowed ensuring maintenance of the retention capacity of PGC column. Intra-assay and inter assay were performed to check the retention time stability using this wash procedure. During intra-assay of 90 injections, retention time was maintained at 6.31 +/-0.07 min for ATP with a RSD of 1.35 %. Inter-assay retention time stability was evaluated on 6 batches (n= 90 injections) and retention time were 6.32 +/-0.07 min with RSD of 1.31 %. We have no obvious explanation of the role of vitamin C, but we suggest that its effect is related to a limitation of the impact of redox reactions, which limits the alteration of interactions with analytes.

The optimal gradient is described in Table 2. An analytical run was started with ascorbic acid 50 mg.L -1 , in water at 0.5 mL.min -1 for 15 min. It was followed by an equilibration time of 5 min with mobile phase A (2 mM ammonium acetate pH 10 in water) and mobile phase B (acetonitrile) 98/2. Then, samples were analysed using gradient elution program as previously described (Table 2). This procedure allowed maintaining retention capacity with shaper peaks.

Other gradient elution were not retained. At this step of the optimisation, we evaluated the transformation during the analysis of ATP to ADP, and AMP. A standard solution at the concentration of 197.2 nM of ATP alone was injected, and ATP, ADP, and AMP were acquired by the current method. Ratios of ATP/ADP and ATP/AMP intensity were calculated and were higher than 100 (Supplemental figure 1). This allowed us to consider the transformation from ATP to ADP, and AMP as negligible. This assay was also performed for ADP, UTP, and UMP. For all these compounds, ratios of intensities were higher than 100 (Supplemental figure 1).

Sample processing optimisation and selection of internal standard

Many biological matrices can be used for nucleotide quantification. Sample preparation was essential to achieve acceptable sensitivity and reproducibility of quantification in complex biological matrices. The common feature of the reported assay was to inhibit the enzymatic degradation of phosphorylated nucleotides. Among these methods, some authors have used the addition of perchloric or trichloroacetic acid or methanol to blood/plasma or to cell matrices [START_REF] Machon | Fully validated assay for the quantification of endogenous nucleoside mono-and triphosphates using online extraction coupled with liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Cohen | Simultaneous analysis of eight nucleoside triphosphates in cell lines by liquid chromatography coupled with tandem mass spectrometry[END_REF][START_REF] Zhu | A simultaneously quantitative method to profiling twenty endogenous nucleosides and nucleotides in cancer cells using UHPLC-MS/MS[END_REF][START_REF] Cortese | Validation of a LC/MSMS method for simultaneous quantification of 9 nucleotides in biological matrices[END_REF][START_REF] Zhang | Strategies for quantitation of endogenous adenine nucleotides in human plasma using novel ion-pair hydrophilic interaction chromatography coupled with tandem mass spectrometry[END_REF][START_REF] Van Moorsel | Differential effects of gemcitabine on ribonucleotide pools of twenty-one solid tumour and leukaemia cell lines[END_REF]. A sonication step can be added to promote cell lysis [START_REF] Chen | A LC-MS/MS method for the analysis of intracellular nucleoside triphosphate levels[END_REF][START_REF] Zhu | A simultaneously quantitative method to profiling twenty endogenous nucleosides and nucleotides in cancer cells using UHPLC-MS/MS[END_REF].

After centrifugation, the supernatants can be injected directly. Some authors carry out an additional liquid/liquid extraction step [START_REF] Canelas | Quantitative evaluation of intracellular metabolite extraction techniques for yeast metabolomics[END_REF][START_REF] Graven | Purine metabolite and energy charge analysis of Trypanosoma brucei cells in different growth phases using an optimized ionpair RP-HPLC/UV for the quantification of adenine and guanine pools[END_REF] or liquid/solid extraction step [START_REF] Cohen | Simultaneous analysis of eight nucleoside triphosphates in cell lines by liquid chromatography coupled with tandem mass spectrometry[END_REF]. For culture media analysis, heating and freezing to -80 °C were described in the sample preparation to block nucleotide metabolism [START_REF] Cohen | Simultaneous analysis of eight nucleoside triphosphates in cell lines by liquid chromatography coupled with tandem mass spectrometry[END_REF].

For the analysis of the cells culture medium, we evaluated three types of extraction: simple evaporation of the supernatant, solid phase extraction and liquid/liquid extraction. For all these tests, 50 µL of supernatant was used and as described in the literature 60 % methanol was added then the extracts were frozen at -80 °C to inhibit ectonucleotidase activity [START_REF] Cohen | Simultaneous analysis of eight nucleoside triphosphates in cell lines by liquid chromatography coupled with tandem mass spectrometry[END_REF][START_REF] Zhang | Strategies for quantitation of endogenous adenine nucleotides in human plasma using novel ion-pair hydrophilic interaction chromatography coupled with tandem mass spectrometry[END_REF][START_REF] Canelas | Quantitative evaluation of intracellular metabolite extraction techniques for yeast metabolomics[END_REF]. The first was simple evaporation of the supernatant/methanol mixture and the recovery of the dry residue by ice water. The second was an SPE performed on Strata TM-X-AW 33 µm (Phenomenex), performed according to the manufacturer's recommendations. SPE columns were conditioned with 1 mL methanol and then were equilibrated with 1 mL water.

Calibration standards were loaded in SPE columns and impurities were washed using 2 mM ammonium acetate followed by 1 mL methanol. Finally, elutions were performed using 500 µL of ammonium hydroxide, two times. The elution solutions have been analysed and did not detect ATP and other analytes in the sample. Wash solutions have also been analysed. This revealed the presence of ATP in the methanol solution. Unfortunately, this procedure, which did not allow the compounds of interest to be retained, was not selected.

Liquid/liquid extraction was tested with different proportions of organic solvents as follows:

Method validation

Linearity

The calibration curves of the targeted analytes were reproduced on six different days. Several regressions and weighting factors were tested. For adenosine, cAMP and UDP, the quadratic regression (Y = ax² + bx + c) without weighting met all predefined criteria. Linear regression model was chosen for the other compounds. Over the concentration range considered, the regression coefficient (R²) of the calibration curves was always greater than 0.996 (n = 6) with calibration samples back-calculated to ± 15 % of the nominal concentration. Figure 2 depicted, the calibration curves, the correlation coefficients and the chromatographic peaks associated with the retention times and areas under curve and intensities at the lower limit of quantification for each compound. For adenosine, AMP, ADP and ATP, data-dependent MS/MS was performed to obtain mass spectra (Supplemental figure 2).

Precision and accuracy

The intra-assay and the inter-assay (n = 6) for both precision and accuracy of the four quality controls for each analyte were within the recommended limits (Table 3). The relative standard deviations of quality controls ranged from 3.14 to 14.98 %. The assessment of the accuracy of the quality controls showed a relative standard deviation of less than ± 15 % (85.55 % -114.93 %) of the target concentration at each level tested. These results indicate that the current method is accurate and reproducible.

Matrix effect

The matrix effect was performed (n = 6) at two concentration levels as described in section 2.4. The matrix factor was calculated by comparing the area under the peak of the analyte of interest from the spiked matrix after extraction and the area under the peak of a pure solution of this analyte at the same concentration. A matrix effect was only observed for UTP at low concentration since the matrix factor was 2.35. The use of nicotamide-D4 as internal standard allowed to correct the matrix effect since the normalised matrix factor was 1.19. As recommended, for all the compounds, at low and high concentrations, the relative standard deviation of the normalised matrix factor was less than 15 % for each compound (2.06 % -14.89 %).

The matrix effect has already been studied in other matrices and has given satisfactory results in accordance with current recommendations (data not shown).

Stability

The stability of the mono-and triphosphate nucleosides extracted from the matrix and maintained for 24 h at 15 °C in the reconstitution solution was acceptable since the responses did not differ by more than ± 15 % from the initial values.

Applicability

After the validation process, the method was applied to quantify nucleotides in cultured cells, the average supernatant, and tissues. Intracellular nucleotide concentrations were adjusted according to the number of cells per well. Intracellular concentrations were expressed in nM per one million of cells.

Human umbilical vein endothelial cells

Analysis of nucleotides and adenosine in human umbilical vein endothelial cells was performed after 2 hours of hypoxia and 2 hours of reoxygenation (Figure 3A, B, C, D, E, F, and G). A significant increase in intracellular concentration of adenosine was observed after 2 hours of hypoxia stress, compared to normoxic control (*p < 0.05, n = 6). A significant decrease of intracellular concentration of UMP after 2 hours of hypoxic stress was observed compared to normoxic control (*p < 0.05, n = 6). A significant decrease of intracellular concentration of AMP, ADP, ATP, UMP, UDP and UTP after 2 hours of hypoxia followed by 2 hours of reoxygenation stress was also observed compared to normoxic control (*p < 0.05, **p < 0.01 n = 6). Compared to 2 hours-hypoxia group (£p < 0.05, ££p < 0.01, n = 6), a significant decrease of intracellular concentration of adenosine, AMP, ADP, ATP and UDP was observed at T240 (after 2h-2h of hypoxia-reoxygenation stress), Analysis of the endothelial cell culture medium was performed at different times during hypoxia and reoxygenation. The results have already been described and showed a significant increase in concentrations of ATP, ADP, AMP and adenosine during hypoxia in the extracellular medium [START_REF] Feliu | Complementary Role of P2 and Adenosine Receptors in ATP Induced-Anti-Apoptotic Effects Against Hypoxic Injury of HUVECs[END_REF]. Compared to control T0 group, Figure 3H also shows a significant increase in UTP and UMP during the first stage of hypoxia in the extracellular compartment (p < 0.05).

It is now well established that a nonlytic release of ATP and other nucleotides occurred in all cell types. A constitutive mechanism, such as exocytosis, is involved in the absence of external stimuli. In response to biochemical or mechanical/physical stimuli, the release is enhanced by the engagement of conductive or transport mechanisms [START_REF] Jacobson | Pharmacology of Purine and Pyrimidine Receptors[END_REF]. A large variety of stimuli, such as hypoxic stress, traumatic shock or certain inflammatory conditions can induce the release of ATP from cells [START_REF] Yegutkin | Nucleotide-and nucleoside-converting ectoenzymes: Important modulators of purinergic signalling cascade[END_REF][START_REF] Gerasimovskaya | Extracellular ATP is an autocrine/paracrine regulator of hypoxia-induced adventitial fibroblast growth. Signaling through extracellular signal-regulated kinase-1/2 and the Egr-1 transcription factor[END_REF][START_REF] Urban | Extracellular ATP attenuates ischemia-induced caspase-3 cleavage in human endothelial cells[END_REF][START_REF] Bodin | Purinergic signalling: ATP release[END_REF]. As in our application, Gerasimovskaya et al. [START_REF] Gerasimovskaya | Extracellular ATP is an autocrine/paracrine regulator of hypoxia-induced adventitial fibroblast growth. Signaling through extracellular signal-regulated kinase-1/2 and the Egr-1 transcription factor[END_REF] and Urban et al. [START_REF] Urban | Extracellular ATP attenuates ischemia-induced caspase-3 cleavage in human endothelial cells[END_REF] reported that hypoxia-induced release of ATP into the extracellular medium. Concerning extracellular concentrations of UTP, UDP and UMP during hypoxia, some authors have already suggested that uracil nucleotides can also be released as an extracellular signalling molecule [START_REF] Yegutkin | Nucleotide-and nucleoside-converting ectoenzymes: Important modulators of purinergic signalling cascade[END_REF].

Purine-converting pathways involved different enzymes, such as phosphodiesterase (NPP), NTPDase1/CD39, 5'-nucleotidase/CD73 (5NT), adenylate kinase-1 (AK1), NDP kinase (NDPK), and adenosine deaminase (ADA) [START_REF] Yegutkin | Nucleotide-and nucleoside-converting ectoenzymes: Important modulators of purinergic signalling cascade[END_REF]. These pathways regulated both ATPgenerating and ATP-consuming. ATP is metabolised into ADP, AMP, and adenosine and finally into inosine. These enzymes are localised in the membrane surface, such as NTPDase1/CD39, or in the cytoplasm or the mitochondria, such as AK1 [START_REF] Yegutkin | Nucleotide-and nucleoside-converting ectoenzymes: Important modulators of purinergic signalling cascade[END_REF][START_REF] Yegutkin | Metabolism of circulating ADP in the bloodstream is mediated via integrated actions of soluble adenylate kinase-1 and NTPDase1/CD39 activities[END_REF]. Our current method allows us to study the regulation of nucleotides and nucleosides in both intracellular and extracellular compartments. Here, we have described the influence of 2 hours-2 hours hypoxia-reoxygenation stress on intracellular and extracellular nucleotide concentrations.

Cardiomyocytes

Isoprenaline is a selective agonist for beta-adrenoceptors relative to alpha-adrenoceptors [START_REF] Alexander | CGTP Collaborators, THE CONCISE GUIDE TO PHARMACOLOGY 2017/18: G proteincoupled receptors[END_REF].

Beta1-adrenoceptors (β1-ARs) are largely expressed in the heart where their activation leads to an increase of heart rate and cardiac contractility. β1-ARs are coupled to Gsα protein able to activate adenylate cyclase. The binding of an agonist to these receptors is translated into physiological response involving adenylate cyclase, leading to an increase of intracellular cAMP concentration [START_REF] Stein | Chapter 11 -β-Adrenergic Receptors[END_REF].

In our study, we proposed to quantify intracellular concentrations of cAMP in cardiomyocytes after stimulation with isoprenaline concentrations ranged 0.1 nM to 1 µM (n = 6 per levels).

Figure 4 illustrates the dose-response curves of intracellular concentrations of cAMP. Our experiment allows determining a pEC50 of 6.666 (95 % confidence intervals: 6.511-6.821, R²: 0.9680) for isoprenaline. Our results are in accordance with previously described in literature ranged 6.6 to 7.0 [START_REF] Alexander | CGTP Collaborators, THE CONCISE GUIDE TO PHARMACOLOGY 2017/18: G proteincoupled receptors[END_REF]. Our method is, therefore, suitable for this kind of application.

Comparisons with related methods

Ion-pairing agents in the mobile phase have been widely used in many publications to achieve satisfactory results [START_REF] Klawitter | Development and validation of an assay for the quantification of 11 nucleotides using LC/LC-electrospray ionization-MS[END_REF][START_REF] Cordell | Quantitative profiling of nucleotides and related phosphate-containing metabolites in cultured mammalian cells by liquid chromatography tandem electrospray mass spectrometry[END_REF][START_REF] Chen | A LC-MS/MS method for the analysis of intracellular nucleoside triphosphate levels[END_REF][START_REF] Cohen | Simultaneous analysis of eight nucleoside triphosphates in cell lines by liquid chromatography coupled with tandem mass spectrometry[END_REF][START_REF] Wang | Simultaneous determination of creatine phosphate, creatine and 12 nucleotides in rat heart by LC-MS/MS[END_REF][START_REF] Zhu | A simultaneously quantitative method to profiling twenty endogenous nucleosides and nucleotides in cancer cells using UHPLC-MS/MS[END_REF][START_REF] Zhang | Strategies for quantitation of endogenous adenine nucleotides in human plasma using novel ion-pair hydrophilic interaction chromatography coupled with tandem mass spectrometry[END_REF]. As the use of these agents induced some disadvantages described above, some studies have proposed their methods without ion-pairing agents [START_REF] Machon | Fully validated assay for the quantification of endogenous nucleoside mono-and triphosphates using online extraction coupled with liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Jiang | Simultaneous determination of adenine nucleotides, creatine phosphate and creatine in rat liver by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry[END_REF][START_REF] Pabst | Nucleotide and nucleotide sugar analysis by liquid chromatography-electrospray ionization-mass spectrometry on surface-conditioned porous graphitic carbon[END_REF][START_REF] Thomas | Quantitation of endogenous nucleoside triphosphates and nucleosides in human cells by liquid chromatography tandem mass spectrometry[END_REF][START_REF] Cortese | Validation of a LC/MSMS method for simultaneous quantification of 9 nucleotides in biological matrices[END_REF][START_REF] He | Simultaneous Quantification of Nucleosides and Nucleotides from Biological Samples[END_REF]. In this field, publications have proposed, as our work, a method using a porous graphitic carbon (PGC) column (Hybercarb, ThermoFisher Scientific, San Jose, CA, USA) [START_REF] Machon | Fully validated assay for the quantification of endogenous nucleoside mono-and triphosphates using online extraction coupled with liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Jiang | Simultaneous determination of adenine nucleotides, creatine phosphate and creatine in rat liver by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry[END_REF][START_REF] Pabst | Nucleotide and nucleotide sugar analysis by liquid chromatography-electrospray ionization-mass spectrometry on surface-conditioned porous graphitic carbon[END_REF]. Cordell et al. proposed the quantification of nucleosides and nucleotides on cells [START_REF] Cordell | Quantitative profiling of nucleotides and related phosphate-containing metabolites in cultured mammalian cells by liquid chromatography tandem electrospray mass spectrometry[END_REF]. Sample pre-treatment was liquid-liquid extraction using -20 °C methanol and hexane. Chromatographic separation was conducted with an ion-paring-C18-reverse-phase column. The calibration range was 0.5-100 µM. Zhu et al. proposed a liquid-liquid extraction of cells using methanol and acetonitrile [START_REF] Zhu | A simultaneously quantitative method to profiling twenty endogenous nucleosides and nucleotides in cancer cells using UHPLC-MS/MS[END_REF]. As in our present study, they used PCG for chromatographic separation. However, Zhu et al. added ion-pairing agent (diethylamine) to improve their sensitivity in particular for nucleotide triphosphates, allowing them to obtain 6 nM for the limit of quantification for ATP, instead of 15.4 nM in our method [START_REF] Zhu | A simultaneously quantitative method to profiling twenty endogenous nucleosides and nucleotides in cancer cells using UHPLC-MS/MS[END_REF]. There are some limitations in our present study that may be considered in future projects.

Nicotinamide D4 was used as internal standard, but the structure of this compound is similar just with part of the structure of the compounds. However, it allowed a good correction of the calibration ranges. Gold-standard isotopes labelled nucleotides, brominated or chlorinated derivatives of nucleotides and nucleosides could be used as internal standards in future.

Finally, retention and separation of these compounds is challenging. The commercial availability of columns, particularly for polar compounds, is constantly increasing. It would be interesting to continue to evaluate the performance of new innovative columns.

Conclusion

In this work, we have described the development and the validation of a method for the quantification of ATP, ADP, cADP-ribose, AMP, cAMP, adenosine, UTP, UDP, and UMP using liquid chromatography coupled with high-resolution mass spectrometry. This method presents a good sensitivity which is compatible with the very low concentration of some nucleotides. Through different applications described in this article, we have demonstrated that our method is appropriate for the quantification of nucleotides in both intracellular and extracellular compartment [START_REF] Reepmeyer | Separation and detection of the isomeric equine conjugated estrogens, equilin sulfate and Δ8,9-dehydroestrone sulfate, by liquid chromatography-electrospray-mass spectrometry using carbon-coated zirconia and porous graphitic carbon stationary phases[END_REF][START_REF] Alexander | CGTP Collaborators, THE CONCISE GUIDE TO PHARMACOLOGY 2017/18: G proteincoupled receptors[END_REF]. We have described an increase of extracellular concentration of ATP, ADP, AMP, adenosine, UTP, UTP, and UMP during the first step of hypoxia. Intracellular concentrations were also described during hypoxia-reoxygenation stress.

Signalisation pathways may be studied with the quantification of cAMP, a secondary intracellular messenger involved in physiological functions. Using a normalised log concentration-effect curves, this method allows the determination of EC50 of isoprenaline for beta-adrenergic receptors in cardiomyocytes.

Conflict of interest

None declared.

  Acquity BEH HILIC (50 x 2.1 mm, 1.7 µm), Acquity HSS T3 (50 x 2.1 mm, 1.8 µm) (Waters, Milford, MA, USA), Uptisphere® ODB (150 x 4.6 mm, 5 µm) (Interchim, Monluçon, France), Syncronis aQ (100 x 2.1 mm, 1.7 µm), Accucore Phenyl-Hexyl (100 x 2.1 mm, 2.6 µm), Hypercarb (150 x 2.1 mm, 5 µm) (ThermoFisher Scientific, San Jose, USA). Due to the high polarity of the compounds, poor separation and insufficient retention time were observed with Acquity HSS T3, Syncronis, HypersilGold, and Accucore Phenyl-Hexyl. The acquity BEH HILIC, Uptisphere columns provided sufficient retention time but were associated with peak residues, poor separation and lack of sensitivity, especially for di-and tri-phosphate compounds. The last column tested and retained was the Hybercarb column, a porous graphitic carbon (PGC) column. The PGC surface is composed of flat sheets of hexagonal carbon atoms in the form of a very large aromatic carbon molecule. The interaction mechanisms are very different from those usually encountered in conventional chromatography. Porous graphitic carbon (PGC) columns can tolerate pH values from 0 to 14 and provide significant retention for targeted compounds. The main disadvantages of the PGC column are a long separation time and a loss of sensitivity for a pH below 10.

  (50/50, V/V). Neither liquid/liquid nor SPE extraction allowed nucleotides and adenosine to be extracted from the culture medium. Only the evaporation of the supernatant recovered with ice water gave satisfactory results.Evaporation and recovery optimisation were performed to achieve good sensitivity as follows: evaporation of 50 µL recovered with 50 µL ice water, 50 µL recovered with 100 µL and 100 µL recovered with 50 µL. The best signal was obtained with 50 µL recovered with 100 µL of ice water.Selection of internal standard:Several internal standards were tested: 5-bromouracil, and 8-bromoadenosine-3'-5'cyclophosphate and nicotinamide-D4. Bromouracil did not allow to obtain satisfactory intensity and therefore was not retained. Calibration ranges were performed with nicotinamide D4 and 8-bromoadenosine-3'-5'cyclophosphate. 8-bromoadenosine-3'-5'cyclophosphate didn't correct the calibration range whereas nicotinamide D4 allowed a satisfactory calibration curve for all compounds. Although the structure of nicotinamide D4 is close to only part of the structure of the compounds, the retention time of nicotinamide-D4, 7.05 min, is close to the compounds of interest. Nicotinamide-D4 will undergo the same matrix effect as the compounds of interest and will allow a better correction of the calibration ranges. Due to these results, nicotinamide-D4 was retained as the internal standard. We have chosen a single internal standard to facilitate the analytical procedure in the field of basic fundamental research.

Figure 1 :

 1 Figure 1: Mobile phase optimisation using 100 µg.L -1 pure solutions of ATP, ADP and AMP

Figure 2 :

 2 Figure 2: Calibration curve with a coefficient of correlation for each compound. For each

Figure 3 :

 3 Figure 3: Analysis of nucleotides and adenosine in human umbilical vein endothelial cells:

Figure 4 :Supplemental figure 1 :Supplemental figure 2 :

 412 Figure 4: Normalised log concentration-effect curves: intracellular concentrations of cAMP

  15.4-1971.7 nM for ATP, 18.3-2340.8 nM for ADP, 22.5-2880.8 nM for AMP, 23.7-3037.6 nM for cAMP, 28.8-1840.5 nM for cADP-ribose, 29.2-3741.9 nM for adenosine, 16.1-2065.5 nM for UTP, 19.3-2474.3 nM for UDP and 24.1-3084.7 nM for UMP. Quality controls at four different concentrations QC1, QC2, QC3 and QC4 were prepared in our laboratory. The calibration (C1, C2, C3, C4, C5 and C6) and QC levels of the samples expressed in µg.L -1 and nM are provided in Table

  Thomas et al. proposed the quantification of nucleosides and triphosphate nucleotides, from human cells using liquid-liquid chromatography[START_REF] Thomas | Quantitation of endogenous nucleoside triphosphates and nucleosides in human cells by liquid chromatography tandem mass spectrometry[END_REF]. In their work, two different chromatographic columns were necessary, one for nucleosides and, an anion exchange column for triphosphate nucleotide. They achieved a limit of quantification of 25 ng.mL -1 for ATP, corresponding to 49 nM. To date, onlyHe et al., proposed as this present study, the quantification of ATP, ADP, AMP, and adenosine without the need of ion-pair agents[START_REF] He | Simultaneous Quantification of Nucleosides and Nucleotides from Biological Samples[END_REF]. Their developed method allowed the simultaneous quantification of 65 nucleosides and nucleotides in human plasma and urine. The extraction of plasma samples was performed using methanol-water. pH was adjusted to 8.5, and then, solid-phase extraction was used to purify extracted nucleosides and nucleotides. HSS T3 column (Waters Corp, MA, USA) was used for chromatographic separation. They achieved a lower limit of quantification of 39.06 nM for adenosine and 2500 nM for ATP[START_REF] He | Simultaneous Quantification of Nucleosides and Nucleotides from Biological Samples[END_REF]. Even if Cortese et al. and Jiang et al. did not quantify adenosine in their studies, it was interesting to relate their study since they obtain an interested lower limit of quantification for ATP of 12.5 fM, and 19.72 nM respectively[START_REF] Jiang | Simultaneous determination of adenine nucleotides, creatine phosphate and creatine in rat liver by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry[END_REF][START_REF] Cortese | Validation of a LC/MSMS method for simultaneous quantification of 9 nucleotides in biological matrices[END_REF]. Using the Hybercarb column, Jiang et al. quantified nucleotides from tissues after a liquid-liquid extraction using methanol and water[START_REF] Jiang | Simultaneous determination of adenine nucleotides, creatine phosphate and creatine in rat liver by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry[END_REF]. The limit of quantification obtained by Jiang et al. for ATP was 19.72 nM is close to that obtained in our study(15.4 nM). compatible with their work. In our study, we achieve retention times of 6.23 min for ATP, 6.21 min for ADP, 6.42 min for AMP, 8.13 min for adenosine, 5.94 min for UTP, 6.00 min for UDP and 6.02 min for UMP. If we compare our results with other studies using PGC columns, Jiang et al. obtained the followed retention time: ATP 7.42 min, ADP 7.33 min, AMP 7.23 min but did not quantified adenosine[START_REF] Jiang | Simultaneous determination of adenine nucleotides, creatine phosphate and creatine in rat liver by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry[END_REF]. During the optimisation phase, we have tested different elution gradients to improve the separation of the compounds.When we obtained a minor better separation of ATP, ADP, and AMP, we could no more obtain a correct retention time for adenosine. So, a compromise was reached to be able to quantify nucleotides and adenosine simultaneously. Like our study, Zhu et al. quantified simultaneously nucleotides and adenosine using a Hypercarb column (100 × 2.1 mm, 5 µm) but they combined to ion-pairing agent in mobile phase[START_REF] Zhu | A simultaneously quantitative method to profiling twenty endogenous nucleosides and nucleotides in cancer cells using UHPLC-MS/MS[END_REF]. They have obtained the followed retention time: ATP 3.8 min, ADP 3.76 min, AMP 3.68 min, adenosine 6.86 min, UTP 1.67 min, UDP 1.47 min, and UMP 1.28 min. Retention times were shorter than ours, particularly

	The retention and separation of these highly polar compounds is a major challenge in the development of analytical methods. In addition, the high structural similarity between these compounds (ATP C10H16N5O13P3, ADP C10H15N5O10P2, AMP C10H14N5O7P, cAMP C10H12N5O6P, cADP-ribose C15H21N5O13P2) also makes difficult the chromatographic separation. With their proper choices of chromatographic conditions, each team tried to reach the best compromise to obtain good retention, correct separation of the different compounds, times were shorter than ours, but the separation of analytes gave satisfactory results with retention times of 2.90 min for ATP, 2.85 for ADP, 4.26 min AMP, 6.22 min for adenosine, and a sensitivity quantified simultaneously nucleosides and adenosine using an HSST3 column [31]. Retention 2.26 min for UDP and 3.22 min for UMP.

for UTP, UDP, and UMP, without a better separation of analytes. If we are interested in analytical techniques using technology other than the PGC, two studies are to be cited: Cortese et al. and He et al.

[START_REF] Cortese | Validation of a LC/MSMS method for simultaneous quantification of 9 nucleotides in biological matrices[END_REF][START_REF] He | Simultaneous Quantification of Nucleosides and Nucleotides from Biological Samples[END_REF]

. Cortese et al. used BEH Amide XBridge for chromatographic separation

[START_REF] Cortese | Validation of a LC/MSMS method for simultaneous quantification of 9 nucleotides in biological matrices[END_REF]

. They also obtained separation of analytes with retention times of 5.081 min for ATP, 4.917 min for ADP, 4.743 min for AMP, 5.192 min for UTP, 4.968 for UDP min and 4.869 min for UMP but adenosine was not quantified. He et al.

Table legends :Table 1 :

 legends1 Levels of both calibrators and quality controls for all targeted compounds expressed both µg.L -1 and nM.

Table 2 :

 2 Mobile phase gradient parameters.

Table 3 :

 3 Intra-assay and inter-assay precision (standard deviation %) and accuracy (% mean compared to target value) for each compound at four levels of concentrations (n = 6).

Table 3 :

 3 Intra-assay and inter-assay precision (standard deviation %) and accuracy (% mean compared to target value) for each compound at four levels of concentrations (n = 6).

	QC1
	Compounds

Table 4 :

 4 Matrix effect at low and high concentration levels (n = 6).

	Low concentration

Acknowledgements:

This work was supported by Reims University Hospital, France.