Carmen Kalalian
email: carmenkalalian@hotmail.com

Gisèle Estelle Roth

El Dib

H J Singh

P K Rao

Abdelkhaleq Chakir
email: abdel.chakir@univ-reims.fr

G El Dib

Product investigation of the gas phase ozonolysis of 1-penten-3-ol, cis-2-penten-1-ol and trans-3-hexen-1-ol

Keywords: Biogenic volatile organic compounds (BVOC), Unsaturated alcohols, Ozonolysis, Products formation yields, Reaction mechanisms

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

To improve our knowledge on the atmospheric fate of organic compounds and their transformation to other secondary pollutants, more information on products formation and reaction mechanisms are needed. Biogenic volatile organic compounds (BVOCs) emitted by vegetation are the main source of VOC emissions in the troposphere [START_REF] Guenther | A global model of natural volatile organic compound emissions[END_REF][START_REF] Kesselmeier | Biogenic volatile organic compound (VOC): An overview on emissions[END_REF][START_REF] Williams | Quantifying the uncertainty in simulating global tropospheric composition due to the variability in global emission estimates of biogenic volatile organic compounds[END_REF]. Other sources, such as the oceans, are considered to be minor sources [START_REF] Broadgate | Seasonal emissions of isoprene and other reactive hydrocarbon gases from the ocean[END_REF][START_REF] Ciuraru | Unravelling new processes at interfaces : photochemical isoprene production at the sea surface[END_REF][START_REF] Yassaa | Evidence for marine production of monoterpenes[END_REF]. Biogenic sources contribute to more than 90% of global VOC emissions where 1150 x 10 12 g of Carbon result from BVOC per year [START_REF] Guenther | A global model of natural volatile organic compound emissions[END_REF]. Such BVOCs mainly include isoprenoids or terpenoids (C 5 H 8 ) n like monoterpenes (n = 2) responsible for plant odors and sesquiterpenes (n = 3). The latter is considered as major products of essential oils stored by plants. BVOCs also include species of other families like alkanes, alkenes, oxygenated volatile organic compounds (OVOC) such as carbonyls, alcohols, esters, ethers and acids. These compounds play a dominant role in the chemistry of the lower troposphere and the atmospheric boundary layer [START_REF] Fuentes | Biogenic Hydrocarbons in the Atmospheric Boundary Layer: A Review[END_REF].

Additionally, certain OVOCs are released from damage occurring in the leaves (grazing, cutting, harvesting, freezing and drying). The main result is C5 and C6 unsaturated alcohols and aldehydes known as "green leaf volatiles" released by plants in response to injury [START_REF] Davis | Rate coefficients for the gas-phase reaction of OH with (Z)-3-hexen-1-ol, 1-penten-3-ol, (E)-2-penten-1-ol, and (E)-2-hexen-1-ol between 243 and 404[END_REF][START_REF] Hatanaka | The biogeneration of green odour by green leaves[END_REF][START_REF] Nakamura | Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both gram-negative and gram-positive bacteria[END_REF]. Their emission depends on temperature, intensity of solar radiation, humidity and other physical and meteorological parameters (Karl et al., 2001b). Among the most widely documented woundinduced emissions are a series of C5 and C6 compounds and their derivatives. These compounds are detected in the atmosphere, especially C6 compounds, with concentrations varying between 0.01 µg (C) / g of dry matter and 1.30 mg (C) / g of dry matter or 1.9 g (C) / m 2 [START_REF] Davis | Rate coefficients for the reaction of OH with (E)-2-pentenal, (E)-2-hexenal, and (E)-2-heptenal[END_REF][START_REF] Davis | Rate coefficients for the gas-phase reaction of OH with (Z)-3-hexen-1-ol, 1-penten-3-ol, (E)-2-penten-1-ol, and (E)-2-hexen-1-ol between 243 and 404[END_REF][START_REF] Fall | Chapter 2 -Biogenic Emissions of Volatile Organic Compounds from Higher Plants A2 -Hewitt, C. Nicholas BT -Reactive Hydrocarbons in the Atmosphere[END_REF][START_REF] Fisher | The biochemical origin of pentenol emissions from wounded leaves[END_REF][START_REF] Fuentes | Biogenic Hydrocarbons in the Atmospheric Boundary Layer: A Review[END_REF][START_REF] Grosjean | Atmospheric chemistry of unsaturated alcohols[END_REF][START_REF] Grosjean | Carbonyl products of the ozone-unsaturated alcohol reaction[END_REF][START_REF] Hatanaka | The biogeneration of green odour by green leaves[END_REF][START_REF] Heiden | Emissions of oxygenated volatile organic compounds from plants part I: Emissions from lipoxygenase activity[END_REF]Karl et al., 2001aKarl et al., , 2001b;;[START_REF] Kirstine | Emissions of volatile organic compounds ( primarily oxygenated species ) from pasture[END_REF][START_REF] Lin | Kinetics and mechanisms of gas phase reactions of hexenols with ozone[END_REF][START_REF] Nakamura | Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both gram-negative and gram-positive bacteria[END_REF][START_REF] Orlando | Rate coefficients and product yields from reaction of OH with 1-penten-3-ol, (Z)-2-penten-1-ol, and allyl alcohol (2-propen-1-ol)[END_REF]. Hexenols are mainly derived from linoleic and linolenic acids, wounding, drying and pathogen attack [START_REF] Heiden | Emissions of oxygenated volatile organic compounds from plants part I: Emissions from lipoxygenase activity[END_REF][START_REF] Papagni | Gas-phase rate coefficients for reactions of NO 3 , OH, O 3 and O( 3 P) with unsaturated alcohols and ethers: Correlations and structure-activity relations (SARs)[END_REF]. Meanwhile, pentenols like 1-penten-3-ol and 2-penten-1-ol can be emitted into the atmosphere from freeze-damaged leaves, with concentrations of the order of ppbv, resulting from the peroxidation of fatty acids or for microbiological protection purpose [START_REF] Davis | Rate coefficients for the gas-phase reaction of OH with (Z)-3-hexen-1-ol, 1-penten-3-ol, (E)-2-penten-1-ol, and (E)-2-hexen-1-ol between 243 and 404[END_REF]Karl et al., 2001b;[START_REF] Orlando | Rate coefficients and product yields from reaction of OH with 1-penten-3-ol, (Z)-2-penten-1-ol, and allyl alcohol (2-propen-1-ol)[END_REF].

Their atmospheric lifetime is influenced by the presence of the C = C double bond which facilitates their oxidation by the photo-oxidants (OH, NO 3 and O 3 ) [START_REF] Davis | Rate coefficients for the gas-phase reaction of OH with (Z)-3-hexen-1-ol, 1-penten-3-ol, (E)-2-penten-1-ol, and (E)-2-hexen-1-ol between 243 and 404[END_REF]. These processes can contribute to the formation of other oxygenated species like carbonyl and carboxylic acids, tropospheric ozone and secondary organic aerosols (SOA) [START_REF] Coates | A comparison of chemical mechanisms using tagged ozone production potential (TOPP) analysis[END_REF][START_REF] Jenkin | Photochemical ozone creation potentials for oxygenated volatile organic compounds: Sensitivity to variations in kinetic and mechanistic parameters[END_REF] which would affect the tropospheric composition as well as the climate. It is therefore important not only to study their reactivity toward these oxidants but also to determine the type and the products yields of the formed compounds.

The oxidation reactions of C5 and C6 unsaturated alcohols with OH radicals has been relatively well studied [START_REF] Davis | Rate coefficients for the gas-phase reaction of OH with (Z)-3-hexen-1-ol, 1-penten-3-ol, (E)-2-penten-1-ol, and (E)-2-hexen-1-ol between 243 and 404[END_REF][START_REF] Gibilisco | OH-initiated degradation of a series of hexenols in the troposphere. Rate coefficients at 298K and 1atm[END_REF][START_REF] Calvé | Kinetic studies of OH and O 3 reactions with allyl and isopropenyl acetate[END_REF][START_REF] Mellouki | Kinetics and mechanisms of the oxidation of oxygenated organic compounds in the gas phase[END_REF][START_REF] Orlando | Rate coefficients and product yields from reaction of OH with 1-penten-3-ol, (Z)-2-penten-1-ol, and allyl alcohol (2-propen-1-ol)[END_REF][START_REF] Papagni | Gas-phase rate coefficients for reactions of NO 3 , OH, O 3 and O( 3 P) with unsaturated alcohols and ethers: Correlations and structure-activity relations (SARs)[END_REF]. However, studies of the atmospheric degradation of these compounds (alcohols > C4) by ozone are still limited to few studies. The rate coefficients of the ozonolysis of the compounds considered during the present investigation have been determined at room temperature and as a function of temperature in previous studies [START_REF] Kalalian | Temperature dependent kinetic study of the gas phase reaction of ozone with 1-penten-3-ol , cis-2penten-1-ol and trans-3-hexen-1-ol : Experimental and theoretical data[END_REF]. The atmospheric lifetime of C5-C6 unsaturated alcohols were calculated and found to vary from few minutes to few days, indicating that their lifetimes were affected by the local chemical composition and the location of their emission sources [START_REF] Grosjean | Rate Constants for the gas-phase reactions of ozone with unsaturated aliphatic alcohols[END_REF][START_REF] Kalalian | Temperature dependent kinetic study of the gas phase reaction of ozone with 1-penten-3-ol , cis-2penten-1-ol and trans-3-hexen-1-ol : Experimental and theoretical data[END_REF][START_REF] Lin | Kinetics and mechanisms of gas phase reactions of hexenols with ozone[END_REF][START_REF] O'dwyer | The gas-phase ozonolysis of 1-penten-3-ol, (Z)-2-penten-1-ol and 1-penten-3-one: Kinetics, products and secondary organic aerosol formation[END_REF]. Moreover, other studies have focused their attention on the determination of the products formed during the ozonolysis reactions of different unsaturated alcohols like 3-buten-1-ol, 2-buten-1-ol, 3-buten-2-ol, 2-methyl-3-buten-2-ol, 2-buten-1,4diol, cis-2-penten-1-ol, 1-penten-3-ol, cis-3-hexen-1-ol, and have established reaction mechanisms [START_REF] Aschmann | Products of the gas-phase reactions of cis-3-hexen-1-ol with OH radicals and O 3[END_REF][START_REF] Grosjean | Atmospheric chemistry of unsaturated alcohols[END_REF][START_REF] Grosjean | Carbonyl products of the ozone-unsaturated alcohol reaction[END_REF][START_REF] O'dwyer | The gas-phase ozonolysis of 1-penten-3-ol, (Z)-2-penten-1-ol and 1-penten-3-one: Kinetics, products and secondary organic aerosol formation[END_REF]. In the studies of [START_REF] Grosjean | Atmospheric chemistry of unsaturated alcohols[END_REF] and [START_REF] Grosjean | Carbonyl products of the ozone-unsaturated alcohol reaction[END_REF], carbonyl products were isolated as 2,4-dinitrophenyl hydrazones by sampling the reaction mix through small C 18 cartridges covered by 2,4-dinitrophenylhydrazine (DNPH) and then analyzed by liquid chromatography. Experiments were performed at room temperature and at atmospheric pressure and at 50% of relative humidity. In addition, O'Dwyer et al.

(2010) determined the primary products of the ozonolysis reaction of 1-penten-3-ol and cis-2penten-1-ol by FTIR and GC-MS at room temperature and atmospheric pressure and at a relative humidity (RH) lower than 1%. To the extent of our knowledge, no mechanistic study of the ozonolysis reaction of trans-3-hexen-1-ol exists in the literature. Thus, in order to improve our understanding on the mechanisms occurring in these processes and enrich the mechanistic databases, further experimental work on products investigation is still required due to discrepancies in some of the experimental products formation yields.

Keeping in view the above facts, in this work, the products of the gas phase ozonolysis of three unsaturated alcohols 1-penten-3-ol (C 5 ) (1P3OL), cis-2-penten-1-ol (C 5 ) (c-2P1OL) and trans-3-hexen-1-ol (C 6 ) (t-3H1OL) were identified using state of the art analytical techniques and their corresponding yields were determined at 298 K and 760 Torr. Attempts have been made to provide plausible reaction mechanisms for the ozonolysis of the targeted alkenols as well. This work will provide complementary information, in terms of products identification and reaction pathways, to previous studies for C5 alcohols [START_REF] Aschmann | Products of the gas-phase reactions of cis-3-hexen-1-ol with OH radicals and O 3[END_REF][START_REF] Grosjean | Atmospheric chemistry of unsaturated alcohols[END_REF][START_REF] Grosjean | Carbonyl products of the ozone-unsaturated alcohol reaction[END_REF][START_REF] O'dwyer | The gas-phase ozonolysis of 1-penten-3-ol, (Z)-2-penten-1-ol and 1-penten-3-one: Kinetics, products and secondary organic aerosol formation[END_REF] and a first reported mechanistic study for trans-3-hexen-1-ol. This information is crucial for the development of reliable atmospheric chemistry models in order to predict the formation of photo-oxidants such as the formation of secondary organic aerosols.

Experimental section

In this work, an Atmospheric Simulation Chamber (ASC) was used. It consisted of a Pyrex reactor (Volume = 63L) connected to a FTIR spectrometer and to a GC-MS. Experiments were carried out at room temperature (T = 298 ± 2 K) and at a pressure of 760 torr in the presence of an excess of cyclohexane used as scavenger of OH radicals. The experimental setup consisted of a triple jacket pyrex reactor transparent to the photolytic light at wavelength greater than 290 nm. This reactor is composed of a multiple reflections cell (length: 200 cm; diameter: 20 cm) with a water flow between the two jackets of the reactor allowing it to maintain the temperature at 298±2 K. The pressure inside the reactor could vary from few mTorr to 1000 Torr. However, in our experimental conditions, the pressure was around 760 Torr. The chamber was equipped with golden mirrors which permitted to vary the optical path, which was set at 56 m in this work and connected to a FTIR spectrometer (Bruker Equinox 55) operating in the range 4000 -400 cm -1 with a resolution of 2 cm -1 .

During an experiment, 40 to 60 IR spectra were collected. Spectra were recorded every 3 minutes and each spectrum constitutes an average of 100 scans to improve the signal to noise ratio. A GC-MS (Thermo Fisher, column: Agilent DB-5MS, 30 m x 0.25 mm x 0.25 µm, carrier gas: Helium) was also used and it consisted of off-line measurements with the use of SPME fibers for the sampling. The different parameters used in GC-MS are summarized in SM-1 supporting materials and further details of the experimental methodology are given in previous publications (Al [START_REF] Rashidi | UV spectra and OH-oxidation kinetics of gaseous phase morpholinic compounds[END_REF][START_REF] Kalalian | Temperature dependent kinetic study of the gas phase reaction of ozone with 1-penten-3-ol , cis-2penten-1-ol and trans-3-hexen-1-ol : Experimental and theoretical data[END_REF][START_REF] Kalalian | Rate coefficients for the gas-phase reaction of ozone with C5 and C6 unsaturated aldehydes[END_REF][START_REF] Laversin | Kinetics of the gasphase reactions of OH radicals and Cl atoms with diethyl ethylphosphonate and triethyl phosphate[END_REF].

Both FTIR and SPME/GC-MS techniques were used to monitor the reagents and products concentrations during experiments. The integrated IR bands for the studied compounds were: 930 -950 cm -1 for 1P3OL; 920-960 cm -1 for c-2P1OL and 939-987 cm -1 for t-3H1OL. These bands correspond to the =C-H deformation and were used to monitor the concentration of the corresponding alcohols. However, analysis in GC-MS were performed using the Total Ion Current (TIC) mode in order to identify all the compounds formed during the ozonolysis reaction.

One should note that the ozonolysis of unsaturated alcohols generates carbonyl, bi-carbonyl and hydroxy-carbonyl compounds. Among these compounds, propanal, glycolaldehyde, acetaldehyde were commercially available, while other compounds such as glyoxal, methylglyoxal and 2-hydroxybutanal were synthesized following the methods given by [START_REF] Horowitz | The UV-VIS absorption cross sections of the α-dicarbonyl compounds: Pyruvic acid, biacetyl and glyoxal[END_REF] and [START_REF] Lockhart | Kinetic study of the OH + glyoxal reaction: Experimental evidence and quantification of direct OH recycling[END_REF], [START_REF] Staffelbach | The UV-visible absorption spectrum and photolysis quantum yields of methylglyoxal[END_REF] and [START_REF] Fessner | Enzymes in organic synthesis, 15. -Short enzymatic synthesis of L-fucose analogs[END_REF], respectively. The purity of the synthesized compounds was checked by GC and FTIR and were further purified by repeated freeze-pump-thaw cycles before use. As for formaldehyde, this compound was prepared by heating paraformaldehyde around 120 °C. A slow flow of nitrogen gas was used to drive the formed formaldehyde into the cell through a cooling trap in order to eliminate water and polymerized products. Formaldehyde concentration was then controlled by UV spectroscopy. The same procedure was also followed for glycolaldehyde dimer to generate gas phase glycoladehyde as a reference standard.

Calibration curves for all the above-mentioned species were made by both FTIR and SPME/GC-MS. Thus, the products of the ozonolysis reactions of targeted alkenols were identified and quantified by comparison with previously established standard spectra in IR and GC-MS.

Prior to any experiment a background check was performed to avoid any carry over from a previous experiment. During each experiment, a concentration of (3-4) × 10 14 molecules cm -3 of alcohols and (9-20) × 10 15 molecules cm -3 of cyclohexane used as OH scavenger, were introduced into the simulation chamber by an air flow after the vaporization of their corresponding liquid standards. Ozone was generated by an ozonizer. The ozone generator used is composed of a double walled pyrex cylinder placed between two copper electrodes and connected to a high frequency generator KIKUSUI-PCCR 500LE, having a single-phase output with a nominal capacity of 500 VA. Ozone is produced by dissociation of the oxygen flowing between the two electrodes. The ozone-oxygen mixture is diluted with dry air at the outlet of the ozonizer and directed to a 12 cm long cylindrical pyrex cell, designed to carry out UV measurements and thus control the quantity of ozone introduced into the simulation chamber. The ozone concentration was monitored at 270 nm at the output of the cell by a CCD camera (Avantes). Ozone was then introduced into the reactor with a continuous flow of 1 mL/min (corresponding to a concentration of the order of 2 x 10 12 molecules cm -3 ).

At the same time, solid phase microextraction (SPME) fibers specifically polydimethylsiloxane / divinylbenzene (PDMS / DVB) were used for sampling. These fibers were previously doped with o-(2, 3, 4, 5, 6 -pentafluorobenzyl) hydroxylamine (PFBHA) [START_REF] Bourdin | Development of SPME on-fiber derivatization for the sampling of formaldehyde and other carbonyl compounds in indoor air[END_REF][START_REF] Reisen | Hydroxyaldehyde products from hydroxyl radical reactions of Z-3-hexen-1-ol and 2-methyl-3-buten-2-ol quantified by SPME and API-MS[END_REF]. The derivatization conditions were taken from [START_REF] Reisen | Hydroxyaldehyde products from hydroxyl radical reactions of Z-3-hexen-1-ol and 2-methyl-3-buten-2-ol quantified by SPME and API-MS[END_REF]. This implies an exposure of the PDMS/DVB fiber for one hour in the head space of a vial containing 4 mL of the aqueous solution of PFBHA (0.4g/L) and agitated with a magnetic stirrer. These conditions ensure a maximum coating of the fiber by the PFBHA and a sufficient quantity of oxime for the detection. The formed carbonyls in the simulation chamber adsorb on the PFBHA-doped fiber and form the corresponding oxime.

Samples were taken approximately every 15 minutes by exposing the doped PDMS/DVB fiber into the simulation chamber for 5 minutes. The fibers were then taken from the chamber and were thermally desorbed at 220° C in the GC-MS injector.

The ozonolysis of the targeted unsaturated alcohols were performed in 3 to 6 replicas. The values of the obtained yields correspond to the average of the determinations made by FTIR and SPME/GC-MS. The uncertainty on the obtained yields corresponds to twice the standard deviation (2σ) and that on the total yield was determined using the error propagation. The carbon balances were also calculated for each time interval of the experiment (every 15 minutes).

The purities of the used chemicals were as follows: 1-penten-3-ol (>98%), cis-2-penten-1-ol (≥96%), trans-3-hexen-1-ol (97%), cyclohexane (≥99%), propanal (≥97%), acetaldehyde (≥99.5%), glycolaldehyde dimer (≥95%), methylglyoxal (40% in H 2 O), paraformaldehyde (> 95%), formic acid (≥95%), propanoic acid (≥99.5%), glyoxal trimer dihydrate (≥95%) and PFBHA (≥98%). All these chemicals were provided by Sigma Aldrich and Santa Cruz and were used without further purification. The gases were supplied by Air Liquide: Air (>99.9999%), O 2 (>99.999%), He (>99.999%).

Results and discussion

Products formation yields

IR spectra at three different reaction times of the reaction of ozone with 1P3OL, c-2P1OL and t-3H1OL are shown in SM-2 in the supporting materials. The approach used to quantify the observed products with the FTIR spectroscopy is based on spectrum subtractions. SM-3 and SM-4 in the supporting materials show the different steps involved in spectra subtractions for 1P3OL and c-2P1OL to determine the concentrations and the formation yields of the observed products. Subtraction of a spectra recorded at time, t of the reaction time from the initial spectrum leads to a spectrum (R), which contains only the corresponding peaks for reaction products. Their formation was confirmed by SPME/GC-MS following the formation of their corresponding oxime. However, for t-3H1OL, the identification of the products was only made by GC-MS due to the lack of a reference for the 3-hydroxypropanal.

Examples of chromatograms for the ozonolysis of 1P3OL, c-2P1OL and t-3H1OL are given in SM-5 in the supporting materials.

Moreover, the molar fractions of the reagents and the identified products were calculated according to:

𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝑓𝑓𝑀𝑀𝑀𝑀𝑓𝑓𝑓𝑓𝑓𝑓𝑀𝑀𝑓𝑓 = 𝐶𝐶 × 𝑇𝑇 × 𝑅𝑅 𝑁𝑁 𝐴𝐴 × 𝑃𝑃
where, C is the concentration of the reagents or the products (molecules cm -3 ), T is the temperature (K), R is the gas constant (R = 8.314 J mol -1 K -1 ), N A is the Avogadro number (N A = 6.022 x 10 23 mol -1 ) and P is the pressure (Pascal).

The temporal evolution profiles of the species involved in the reaction processes are presented in Figure 1. All identified products belong to the family of aldehydes and hydroxyaldehydes.

Figure 1 shows that the concentrations of the identified products are almost stable after 70 minutes of reaction time. Beyond this time, a slight consumption is observed for hydroxycarbonyls and bi-carbonyls; however, a slight increase is observed for glycolaldehyde and propanal for the reaction of c-2P1OL. These variations remain included in the molar fraction uncertainty interval.

The carbon balance was also determined for each time interval of the experiment according to:

Carbon balance = ∑(molar fraction of species at time 𝑓𝑓 × species carbon atom number ) molar fraction of reagents at time 𝑓𝑓 0 × reagent carbon atom number × 100

Results plotted in Figure 1 show that a carbon deficit was observed when the rate of consumption of the starting reagent exceeded 30%. At the end of the reaction the carbon deficit can reach up to 50% for 1P3OL, 65% for c-2P1OL and 60% for t-3H1OL. This is due to the possible formation of additional products in the reactor that could not be detected or identified by the used analytical techniques on account of the limitation associated to the derivatization agent, lack in reference standards and the detection limit of the instruments.

The incomplete carbon balance might also result from an interaction between the gas and the aerosol phase. In fact, in the study of O'Dwyer et al. ( 2010), the formation of secondary organic aerosol (SOA) for the ozonolysis of unsaturated alcohols (1-penten-3-ol and cis-2penten-1ol) was observed with yields ranging from 0.13 -0.17. However, we do not have the means to do these SOA measurements in our laboratory.

The formation yields of primary (2-hydroxybutanal and formaldehyde for 1P3OL, glycolaldehyde and propanal for c-2P1OL, 3-hydroxypropanal and propanal for t-3H1OL)

and secondary products (propanal and acetaldehyde for 1P3OL, acetaldehyde, 2hydroxypropanal, and methylglyoxal for c-2P1OL, and glycolaldehyde, methylglyoxal and acetaldehyde for t-3H1OL) for the reaction of O 3 with 1P3OL, c-2P1OL and t-3H1OL were determined by plotting their concentration as a function of that of the alcohol for increasing times by FTIR and SPME/GC-MS (Figures 234). The formation yields correspond to the slopes of the plotted curves. The average yields obtained by both techniques are summarized in Table 1. The formation yields of primary carbonyl exceed 100% (108±16 for 1-penten-3-ol and 106±13 for trans-3-hexen-1-ol respectively), as the formed primary carbonyls can also be produced via the reactions of Criegee intermediates.

Uncertainty on the average yields are twice the standard deviation. This uncertainty originated mainly from systematic errors due to the used analytical techniques to monitor and quantify the identified compounds. For FTIR analysis, uncertainty on yield is mainly due to errors related to spectra subtraction as well as the area integration of spectroscopic peaks corresponding to both the reagent and the identified products. To reduce this error, more than 40 IR spectra were collected during an experiment and each spectrum constituted an average of 100 scans. For GC/MS analysis, errors are related to chromatographic peak's integration and standard calibration curves. To minimize this error, preliminary tests were performed to optimise SPME-GC/MS method. In addition, experiments were performed in 3-6 replicas.

The yields obtained by both techniques are in good agreement with a difference that do not exceed 20-25%. However, the obtained results by FTIR have higher uncertainties related mainly to peak substractions, which are systematic errors that cannot be avoided. As expected, large error (more than 30%) is mainly observed for species with low concentrations.

These yields were compared to previous literature studies for 1P3OL and c-2P1OL [START_REF] Grosjean | Carbonyl products of the ozone-unsaturated alcohol reaction[END_REF][START_REF] O'dwyer | The gas-phase ozonolysis of 1-penten-3-ol, (Z)-2-penten-1-ol and 1-penten-3-one: Kinetics, products and secondary organic aerosol formation[END_REF]. As for the ozonolysis of t-3H1OL, to the best of our knowledge, no study exists in the literature. The different experimental conditions of this current study and those of the literature are listed in SM-6 in the supporting materials.

The major products identified during the ozonolysis of 1-penten-3-ol were formaldehyde (62 ± 12)% and 2-hydroxybutanal (46 ± 10)% (Table 1). For 2-hydroxybutanal yields, a 35%

discrepancy is observed when compared with the values obtained by Grosjean and Grosjean, As for the ozonolysis of cis-2-penten-1-ol, the major products identified and quantified were glycolaldehyde (56 ± 14)% and propanal (40 ± 10)% as listed in Table 1. For glycolaldehyde yields, 5% and 23% discrepancies are obtained with the values obtained by Grosjean and[START_REF] Grosjean | Carbonyl products of the ozone-unsaturated alcohol reaction[END_REF][START_REF] O'dwyer | The gas-phase ozonolysis of 1-penten-3-ol, (Z)-2-penten-1-ol and 1-penten-3-one: Kinetics, products and secondary organic aerosol formation[END_REF], respectively. As for propanal yields, 28% and 3% discrepancies are observed with the values of [START_REF] Grosjean | Carbonyl products of the ozone-unsaturated alcohol reaction[END_REF] and O'Dwyer et al. ( 2010) respectively. The experimental data obtained for the main products (propanal and glycolaldehyde) are in good agreement with those determined by Grosjean and[START_REF] Grosjean | Carbonyl products of the ozone-unsaturated alcohol reaction[END_REF][START_REF] O'dwyer | The gas-phase ozonolysis of 1-penten-3-ol, (Z)-2-penten-1-ol and 1-penten-3-one: Kinetics, products and secondary organic aerosol formation[END_REF] with an average difference of 17% and 13%, in their respective yields. Other compounds such as 2-hydroxypropanal, methylglyoxal and acetaldehyde have also been observed with the percentage yields of (10 ± 2)%, (3±1)% and (13 ± 5)%, respectively. Traces of formaldehyde were also detected but could not be quantified. In their study of ozonolysis of c-2P1OL, O'Dwyer et al. ( 2010) have only identified acetaldehyde, whereas [START_REF] Grosjean | Carbonyl products of the ozone-unsaturated alcohol reaction[END_REF] identified and quantified acetaldehyde, methylglyoxal and formaldehyde. It should be noted that [START_REF] Grosjean | Carbonyl products of the ozone-unsaturated alcohol reaction[END_REF] assumed that glycolaldehyde, hydroxycarbonyls (2-hydroxypropanal and 2hydroxybutanal) reacted with the acidic DNPH to form the DNPH derivatives of the corresponding di-carbonyls, namely glyoxal, methylglyoxal and 2-oxobutanal. Thus, the 2hydroxypropanal was measured as methylglyoxal-DNPH using the available reference standard.

It should be noted that, relative humidity may affect the products yields during the ozonolysis of unsaturated compounds. In fact several studies, concerning the ozonolysis of alkenes, have

shown that the slightly different amounts of water vapour present in the chambers could affect the reaction pathways of the Criegee intermediates and thus alter product distributions and the generation of OH radicals [START_REF] Hasson | Production of stabilized Criegee intermediates and peroxides in the gas phase ozonolysis of alkenes 2. Asymmetric and biogenic alkenes[END_REF][START_REF] O'dwyer | The gas-phase ozonolysis of 1-penten-3-ol, (Z)-2-penten-1-ol and 1-penten-3-one: Kinetics, products and secondary organic aerosol formation[END_REF][START_REF] Wegener | Simulation chamber investigation of the reactions of ozone with short-chained alkenes[END_REF].

Indeed, the humidity can accelerate the formation of organic hydroperoxides via the hydroperoxide channels. Thus, more experiments for the ozonolysis of other unsaturated compounds, under different relative humidities are still needed.

Hydroxyl group effect on primary carbonyl formation yields

To better understand the effect of a hydroxyl substituent on primary carbonyl formation yields, the formation yields of the products resulting from the ozonolysis of 1P3OL, c-2P1OL and t-3H1OLwere compared to those of their homologous alkenes (Table 2). 1P3OL, c-2P1OL and t-3H1OL were compared to 1-pentene, cis-2-pentene and trans-3-hexene, respectively. These alkenes have the same geometric configuration and the same position of the double bond as that present in the targeted alcohols. The ozonolysis of 1-pentene was performed by [START_REF] Atkinson | Products of the gas phase reactions of O 3 with alkenes[END_REF] in a 7900 L Teflon chamber at 296 ± 2 K and 740 Torr total pressure of purified air at ~5% relative humidity and in the presence of OH scavenger (cyclohexane or n-octane) using FTIR spectroscopy and GC-FID. The ozonolysis of cis-2 pentene and trans-3-hexene were carried out by [START_REF] Grosjean | Carbonyl products of the gas-phase reaction of ozone with simple alkenes[END_REF] and [START_REF] Grosjean | The gas phase reaction of unsaturated oxygenates with ozone: Carbonyl products and comparison with the alkene-ozone reaction[END_REF] in a 3.7-3.9 m 3 Teflon film chambers covered with black plastic. The experiments were performed in the presence of cyclohexane as OH scavenger, at T=286-296K, at atmospheric pressure and at a relative humidity of 55 ± 10% using C18 cartridges coated with 2,4-dinitrophenylhydrazine (DNPH) analyzed by liquid chromatography.

The branching ratio between the two potential routes for the ozonide to decompose seems to be similar between the alcohols and their respective alkenes. This behavior has been also observed for other mono-and di-substituted unsaturated alcohols, carbonyl compounds, ethers and esters [START_REF] Grosjean | Carbonyl products of the gas-phase reaction of ozone with simple alkenes[END_REF]: no influence of the presence of oxygenated groups on the branching ratios was observed. This similarity between alkenes and oxygenated compounds such as alcohol, ether, carbonyl and ester show that the ozone addition to the C=C double bond forms an ozonide and its decomposition follow the same route irrespective of the structure of the compound. However, concerning the reactivity, the presence of functional groups influences the kinetics of the ozonolysis in the gas phase where alkenes, unsaturated ethers and unsaturated alcohols react more rapidly with ozone than aldehydes, ketones and unsaturated esters [START_REF] Grosjean | Carbonyl products of the gas-phase reaction of ozone with simple alkenes[END_REF].

3.3.Mechanistic schemes

As with olefinic compounds, the reaction between unsaturated alcohols and ozone starts by an electrophilic addition of ozone to the C=C double bond to form a primary ozonide. The latter is not stable and cleaves simultaneously to give two primary carbonyl compounds and two intermediates called the "Criegee" intermediate [START_REF] Atkinson | Atmospheric Degradation of volatile organic compounds[END_REF]. Based on the identified products in IR and GC-MS analysis, and the available literature for other unsaturated compounds [START_REF] Atkinson | Gas-phase degradation of organic compunds in the troposphere[END_REF][START_REF] Fessner | Enzymes in organic synthesis, 15. -Short enzymatic synthesis of L-fucose analogs[END_REF][START_REF] Johnson | The gas-phase ozonolysis of unsaturated volatile organic compounds in the troposphere[END_REF][START_REF] O'dwyer | The gas-phase ozonolysis of 1-penten-3-ol, (Z)-2-penten-1-ol and 1-penten-3-one: Kinetics, products and secondary organic aerosol formation[END_REF] , detailed mechanisms of the ozonolysis of 1P3OL, c-2P1OL and t-3H1OL are established (Figure 5 (a-c)).

The total yields of the primary products of the O 3 -reaction with 1-penten-3-ol, cis-2-penten-1ol and trans-3-hexen-1-ol are (108±16) %, (96±17) %, and (106±13) %, respectively.

The products formation yields summarized in Table 1 show that there is no remarkable preference between the evolution pathways of the Criegee bi-radical. In addition, the total yields of the primary products exceed 100%. These results indicate that the observed yields include the formation of these products by both primary (ozonide reactions) and secondary reactions (CI reactions). Therefore, it is not easy to extract the preferential reaction pathway that should be followed by both ozonide and Criegee intermediates.

The Criegee, H 2 C°OO° is produced during the ozonolysis of 1P3OL. Its decomposition leads to the formation of other stable species such as HCHO, CO 2 , CO, H 2 O, H 2 , OH and HC(O)OH [START_REF] Johnson | The gas-phase ozonolysis of unsaturated volatile organic compounds in the troposphere[END_REF]. Determination of H 2 O and CO 2 yields was not attempted due to the interference of the background levels of these compounds in the reaction chamber because part of the beam was exposed to air within the compartment. (iii) Reaction with HCHO to form formic acid HCOOH However, no trace of formic acid was observed in FTIR or GC-MS. This might be linked either to the very low concentration of formed formic acid (under the detection limit of the instrument mainly FTIR) and to the fact that the experiments were conducted at a relative humidity < 1%. Moreover, we assumed that the detected formaldehyde could not result from the cited reactions as the study was conducted under dry condition (RH< 1%).

The Criegee, CH 3 CH 2 CHOHC°HOO° formed during the ozonolysis of 1P3OL can evolve mainly to form 2-hydroxybutanal (46±10)%, formaldehyde (62±12)%), propanal (12±4)% and acetaldehyde (10±4)% (Figure 5a) [START_REF] Grosjean | Carbonyl products of the ozone-unsaturated alcohol reaction[END_REF][START_REF] O'dwyer | The gas-phase ozonolysis of 1-penten-3-ol, (Z)-2-penten-1-ol and 1-penten-3-one: Kinetics, products and secondary organic aerosol formation[END_REF].

The pathway (c') corresponding to the formation of 2-hydroxybutanal and an oxygen atom O

( 3 P) is considered as a minor pathway according to [START_REF] Fessner | Enzymes in organic synthesis, 15. -Short enzymatic synthesis of L-fucose analogs[END_REF]. Propanal has also been identified by [START_REF] O'dwyer | The gas-phase ozonolysis of 1-penten-3-ol, (Z)-2-penten-1-ol and 1-penten-3-one: Kinetics, products and secondary organic aerosol formation[END_REF] and [START_REF] Grosjean | Carbonyl products of the ozone-unsaturated alcohol reaction[END_REF] who proposed that propanal could be produced from the unimolecular decomposition of excited Criegee, as shown in Figure 5a. In addition, no chromatographic peak or band corresponding to propanoic acid and 2-oxobutanal (lack of references) has been identified.

The Criegee, CH 3 CH 2 CH°OO° formed during the reaction of O 3 with c-2P1OL and t-3H1OL [START_REF] Aschmann | Products of the gas-phase reactions of cis-3-hexen-1-ol with OH radicals and O 3[END_REF][START_REF] Grosjean | Atmospheric chemistry of unsaturated alcohols[END_REF][START_REF] Grosjean | Carbonyl products of the ozone-unsaturated alcohol reaction[END_REF][START_REF] O'dwyer | The gas-phase ozonolysis of 1-penten-3-ol, (Z)-2-penten-1-ol and 1-penten-3-one: Kinetics, products and secondary organic aerosol formation[END_REF] can evolve in five different pathways (Figures 5b and5c):

(i) Production of an oxygen atom O ( 3 P) and propanal. This route is minor according to [START_REF] Fessner | Enzymes in organic synthesis, 15. -Short enzymatic synthesis of L-fucose analogs[END_REF].

(ii) Formation of OH radical and CH 3 CH 2 C°O. The latter can react with O 2 to give a peroxypropenyl, which decomposes to form CO 2 and CH 3 CH 2 °. With an excess of O 2 , the ethyl radical leads to the formation of acetaldehyde quantified by SPME/GC-MS with a yield of (13 ± 5)% for c-2P1OL and (30±4)% for t-3-H1OL.

(iii) Formation of ethane and CO 2 . It was not possible to detect ethane by the used analytical techniques.

(iv) Formation of the hydroperoxyde CH 3 CH=CHOOH that can decompose in four different pathways as shown in Figures 5 (b-c). 2-Hydroxypropanal has been identified by FTIR in the case of c-2P1OL with a yield of (10±2) %. Acetaldehyde can also result from this way where it was quantified with a yield of (13 ± 5) % for c-2P1OL and (30±4) % for t-3H1OL. In addition to the above-mentioned compound, methylglyoxal was identified with a yield of (8±3) % for t-3H1OL and (3±1) % for c-2P1OL. This is in accord with the results obtained by [START_REF] Grosjean | Carbonyl products of the ozone-unsaturated alcohol reaction[END_REF] in the study of the ozonolysis of c-2P1OL, where acetaldehyde and methylglyoxal were observed.

(v) Formation of propanoic acid. This was observed neither in FTIR nor in SPME/GC-MS.

The bi-radical HOCH 2 CH°OO°, formed during the ozonolysis of c-2P1OL, can evolve into three different pathways (Figure 5b). However, apart from glycolaldehyde, traces of formaldehyde were observed but not quantified in GC-MS.

The bi-radical OHCH 3 CH 2 C°HOO°, formed during the ozonolysis of t-3H1OL [START_REF] Aschmann | Products of the gas-phase reactions of cis-3-hexen-1-ol with OH radicals and O 3[END_REF][START_REF] Grosjean | Atmospheric chemistry of unsaturated alcohols[END_REF] evolves in five different routes (Figure 5c):

(i) Formation of 3-hydroxypropanal and atomic oxygen. This is a minor pathway according to [START_REF] Fessner | Enzymes in organic synthesis, 15. -Short enzymatic synthesis of L-fucose analogs[END_REF]. (iii) Formation of OH radicals and OHCH 2 CH 2 CO°. In the presence of oxygen, the latter, induce the formation of glycolaldehyde quantified in GC-MS with a yield of (16 ± 4) %.

(iv) Formation of CO 2 , HO 2 and glycolaldehyde. Glycolaldehyde was identified and quantified in GC / MS with a yield of (16 ± 4) %.

(v) Formation of OHCH 2 CH 2 C(O)OH. This compound was not observed with the existing analytical techniques used during the present investigation due to lack of references.

Conclusion

Reaction products and mechanisms of the gas phase reaction of ozone with three unsaturated alcohols (1-penten-3-ol, cis-2-penten-1-ol and trans-3-hexen-1-ol) have been determined at room temperature and atmospheric pressure in an atmospheric simulation chamber. Both the FTIR and the GC-MS techniques were used to monitor the concentrations of the reagents and the formed products. This study shows that their ozonolysis leads to the formation of carbonyl and hydroxylcarbonyl products. These multifunctional species contribute to the increase of the oxidizing capacity of the atmosphere (HO x formation, SOA).

Primary products of the ozonolysis of 1P3OL, c-2P1OL and t-3H1OL are found to be 2hydroxybutanal and formaldehyde, glycolaldehyde and propanal and 3-hydroxypropanal and propanal, respectively. The total formation yields of these products are around 100%. Some secondary products have also been detected and quantified. The yields of primary and secondary products obtained in this work are close to those found in the literature. No preference between the primary ozonide decomposition pathways was found in the ozonolysis of the three unsaturated alcohols undertaken during the present study. Detailed mechanisms for the ozonolysis of the targeted compounds have been developed based on our observations and those available in the literature. a: [START_REF] Atkinson | Products of the gas phase reactions of O 3 with alkenes[END_REF] ; b : Grosjean andGrosjean, (1997), c : Grosjean et al. (1996)

  , whereas it agrees well with the values reported by[START_REF] O'dwyer | The gas-phase ozonolysis of 1-penten-3-ol, (Z)-2-penten-1-ol and 1-penten-3-one: Kinetics, products and secondary organic aerosol formation[END_REF]. As for formaldehyde yields, 45% and 21% discrepencies are observed with the yields determined byGrosjean and[START_REF] Grosjean | Carbonyl products of the ozone-unsaturated alcohol reaction[END_REF][START_REF] O'dwyer | The gas-phase ozonolysis of 1-penten-3-ol, (Z)-2-penten-1-ol and 1-penten-3-one: Kinetics, products and secondary organic aerosol formation[END_REF], respectively. Our results are in good agreement with those of O'Dwyer et al. (2010) who conducted their study at the same relative humidity (RH<1%) as was kept in the present study. The substantial discrepancy with the results of[START_REF] Grosjean | Carbonyl products of the ozone-unsaturated alcohol reaction[END_REF] might be linked to the different relative humidity conditions used. Minor products such as propanal and acetaldehyde have been identified and quantified with yields of (12 ± 4)% and (10 ± 4)% respectively. Acetaldehyde was not quantified byGrosjean and[START_REF] Grosjean | Carbonyl products of the ozone-unsaturated alcohol reaction[END_REF][START_REF] O'dwyer | The gas-phase ozonolysis of 1-penten-3-ol, (Z)-2-penten-1-ol and 1-penten-3-one: Kinetics, products and secondary organic aerosol formation[END_REF]. Formic acid was observed but not quantified by[START_REF] O'dwyer | The gas-phase ozonolysis of 1-penten-3-ol, (Z)-2-penten-1-ol and 1-penten-3-one: Kinetics, products and secondary organic aerosol formation[END_REF]. However, it was not detected in this study.

  OHCH 2 CH = CHOOH. The decomposition of this hydroperoxide may follow 5 different routes as shown in Figure 5c. However, only glycolaldehyde is observed and quantified in GC-MS with a yield of (16 ± 4) %.

Figure 1 :

 1 Figure 1: Carbon balance and temporal evolution of 1-penten-3-ol (a), cis-2-penten-1-ol (b)and trans-3-hexen-1-ol (c) and the formed products identified by SPME/GC-MS and by FTIR during their ozonolysis. Solid lines correspond to the points adjustments using a polynomial fit (R 2 >0.9).

Figure 2 :

 2 Figure 2: Formation yields for primary and secondary products observed during the ozonolysis of 1P3OL obtained by SPME/ GC-MS (a) and by FTIR (b).

Figure 3 :

 3 Figure 3: Formation yields for primary and secondary products observed during the ozonolysis of c-2P1OL obtained by SPME/GC -MS (a) and by FTIR (b).

Figure 4 :

 4 Figure 4: Formation yields for primary and secondary products observed during the ozonolysis of t-3H1OL obtained by SPME/GC -MS.

Figure 5 :

 5 Figure 5: Proposed reaction scheme for the gas phase ozonolysis of 1 penten-3-ol (a), cis-2-penten-1-ol (b) and trans-3-hexen-1-ol (c). The compounds framed with solid lines are detected in the present study whereas the one in the dotted frame corresponds to secondary pathways of the primary products.

Table 1 :

 1 Comparison of the products identified and quantified during the ozonolysis of 1-penten-3-ol, cis-2-penten-1-ol and trans-3-hexen-1-ol. :[START_REF] Grosjean | Carbonyl products of the ozone-unsaturated alcohol reaction[END_REF] ; b : O'Dwyer et al. (2010) ; 1 : detected in this work by FTIR; 2 : detected in this work by GC-MS, n.i..: not identified

	Unsaturated alcohol 1-Penten-3-ol	Identified products 2-Hydroxybutanal 1,2 Formaldehyde 1,2 Propanal 2 Acetaldehyde 2	Average yields-this work (%) 46±10 62±12 12±4 10±4	Literature 30±5 a 46±3 b 34±4 a 49±2 b 12±1 a 15±2 b n.i. a, b n.i. a
	cis-2-Penten-1-ol	Formic acid Glycolaldehyde 1,2 Propanal 1,2 Acetaldehyde 2 2-Hydroxypropanal 1 Formaldehyde 2 Methylglyoxal	-56±14 40±10 13±5 10±2 Identified but not quantified 3±1-	Identified but not quantified b 53±4 a 43±4 b 51±2 a 39±3 b 8±1 a Identified but not quantified b n.i. a, b 7±2 a n.i. b 4±1 a n.i. b
		3-Hydroxypropanal 2	43±10
		Propanal 2	63±8
	trans-3-Hexen-1-ol	Glycolaldehyde 2	16±4
		Acetaldehyde 2	30±4
		Methylglyoxal 2	8±3

a

Table 2 :

 2 Comparison of the primary products formation yields (R) of unsaturated alcohols with their homologous alkenes.

	Compounds	Primary products	R (%)	Homologous alkenes	Primary products	R (%)
	1-Penten-3-ol	Formaldehyde 62±12	1-Pentene	Formaldehyde 60±6 a
	cis-2-Penten-1-ol	Propanal	40±10	cis-2-Pentene	Propanal	51±3 b
	trans-3-Hexen-1-ol	Propanal	63±8	trans-3-Hexene	Propanal	101±5 c

Acknowledgment

The authors thank the INSU-LEFE CHAT program for the financial support.


domain: chim sdv

text:
Annelise Genoux

Thibaut Duparc

Jean-Bernard Ruidavets

Cécile Ingueneau

Souad Najib

Jean Ferrières

Bertrand Perret

Mikaël Croyal
email: mikael.croyal@univ-nantes.fr

Laurent O Martinez
email: laurent.martinez@inserm.fr

A reference measurement of circulating ATPase inhibitory factor 1 (IF1) in humans by LC-MS/MS: Comparison with conventional ELISA

Keywords: AcN, acetonitrile, CHD, coronary heart disease, CI, confidence interval, CV, coefficient of variation, CVD, cardiovascular diseases, EDTA, thylenediaminetetraacetic acid, electrospray ionization, ESI, ELISA, enzyme-linked immunosorbent assay, FA, formic acid, HDL, high-density lipoprotein, IF1, inhibitory factor 1, LC-MS/MS, liquid chromatography-tandem mass spectrometry method, MeOH, methanol, multiple reaction monitoring, MRM, QC, quality control, SPE, solid-phase extraction, TFA, trifluoroacetic acid max 6): ATPase inhibitory factor 1, mass spectrometry, clinical biomarker, cardiovascular disease, assay development

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Despite many advances in therapeutics, cardiovascular diseases (CVD) remain the leading cause of deaths worldwide. Moreover, the main objective of current management guidelines for CVD is improving the prognosis in high-risk populations. Therefore, there is a critical need to determine novel non-invasive biomarkers to better predict the risk of CVD in the most at-risk patients.

Inhibitory Factor 1 (IF1) is a nuclear-encoded protein known to inhibit the ATP hydrolysis activity of both mitochondrial and plasma membrane ATP synthase. Consequently, IF1 prevents cell death in ischemic conditions and blocks endocytosis of high-density lipoprotein (HDL) by the liver [START_REF] Lippe | Mitochondrial and cellsurface F0F1ATPsynthase in innate and acquired cardioprotection[END_REF][START_REF] Martinez | Ectopic beta-chain of ATP synthase is an apolipoprotein A-I receptor in hepatic HDL endocytosis[END_REF]. In recent studies, IF1 was unexpectedly detected in the systemic circulation in humans. Moreover, in clinical studies, circulating IF1 was found to be an independent determinant of HDL-Cholesterol (HDL-C) levels and its mean concentration was lower in coronary heart disease (CHD) patients than in paired control individuals (430 ng/mL and 530 ng/mL, respectively; p <0.001). Later, IF1 levels were found to independently and negatively associate with mortality in CHD patients [START_REF] Genoux | Mitochondrial Inhibitory Factor 1 (IF1) Is Present in Human Serum and Is Positively Correlated with HDL-Cholesterol[END_REF][START_REF] Genoux | Serum IF1 concentration is independently associated to HDL levels and to coronary heart disease: the GENES study[END_REF][START_REF] Genoux | Serum levels of mitochondrial inhibitory factor 1 are independently associated with long-term prognosis in coronary artery disease: the GENES Study[END_REF]. Recently, higher levels of circulating IF1 were also reported to be significantly associated with greater muscle mass [START_REF] Lee | ATP synthase inhibitory factor 1 (IF1), a novel myokine, regulates glucose metabolism by AMPK and Akt dual pathways[END_REF].

These studies support the clinical potential of IF1 measurement for the assessment of cardiovascular risk in high-risk populations.

Further, the aforementioned studies have been performed using competitive enzyme immunoassays with different polyclonal anti-IF1 antibodies and recombinant IF1 protein [START_REF] Genoux | Mitochondrial Inhibitory Factor 1 (IF1) Is Present in Human Serum and Is Positively Correlated with HDL-Cholesterol[END_REF][START_REF] Lee | ATP synthase inhibitory factor 1 (IF1), a novel myokine, regulates glucose metabolism by AMPK and Akt dual pathways[END_REF]. However, such immunoassays are limited by batch size and batch-to-batch variability regarding both polyclonal antibodies and recombinant proteins batches. Thus, the use of a technique that will not depend on reagents' availability and variability and will enable to establish a reference measurement of circulating IF1 for clinical practice is warranted. In this respect, enzymatic proteolysis of proteins by endoproteases, post-proteolysis concentration of peptides by solid-phase extraction (SPE) and subsequent targeted analysis of signature peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS), has been recently demonstrated as a powerful method to quantify lowabundance plasma proteins [START_REF] Croyal | Plasma PCSK9 measurement by liquid chromatography-Tandem mass spectrometry and comparison with conventional ELISA[END_REF]. The aim of the present study was to develop and validate such an LC-MS/MS assay, enabling the quantification of circulating IF1 and its comparison to the traditional enzyme-linked immunosorbent assay (ELISA) approach.

Materials and Methods

Chemical and reagents

UPLC/MS-grade acetonitrile (AcN), water, methanol (MeOH), trifluoroacetic acid (TFA) and formic acid (FA) were purchased from Biosolve (Valkenswaard, Netherlands). Samples were prepared with the ProteinWorks TM eXpress kit (Waters Corporation, Milford, MA, USA), according to the manufacturer's instructions and consisting in ammonium bicarbonate (50 mM, pH 8) as the digestion buffer, RapidGest (7 mg/mL in digestion buffer) as the detergent, dithiothreitol (70 mM in digestion buffer) as the reducing agent, iodoacetamide (142 mM in digestion buffer) as the alkylation agent, trypsin (7 mg/mL in HCl 1 mM) as the endoprotease, and 20% TFA as the inactivating agent. Moreover, the protein sequence of IF1 was BLAST searched using the UNIPROT tool (www.uniprot.org), and theoretical peptides were searched using ExPASy (http://web.expasy.org/peptide_mass) [START_REF] Croyal | Multiplexed peptide analysis for kinetic measurements of major human apolipoproteins by LC/MS/MS[END_REF]. Synthetic labelled and unlabelled proteotypic peptides (Table 1) were provided by Thermo Scientific Biopolymers (Darmstadt, Germany) at purity > 99%. Stock solutions (1 mM) were prepared in 50% AcN containing 0.1% FA and stored at -20 °C until use. The mature human recombinant IF1 protein was chemically synthesized by GenScript (Piscataway, NJ, USA) at >80% purity. The human IF1 stock solution (1 mg/mL) was prepared in ultra-pure water and stored at -20 °C until use.

Study samples

Ethylenediaminetetraacetic acid (EDTA) plasma and serum samples were obtained from 204 male participants from the case-control GENES (Génétique et Environnement en Europe du Sud) study [START_REF] Genoux | Serum IF1 concentration is independently associated to HDL levels and to coronary heart disease: the GENES study[END_REF].

The study protocol was approved by the local ethics committee (CCPPRB, Toulouse/Sud-Ouest, file #1-99-48, Feb 2000), with all patients having provided written informed consent. The sample collection has been declared as DC-2008-463 #1 to the Ministry of Research and to the Regional Health authority [START_REF] Genoux | Serum IF1 concentration is independently associated to HDL levels and to coronary heart disease: the GENES study[END_REF]. As previously described [START_REF] Genoux | Serum IF1 concentration is independently associated to HDL levels and to coronary heart disease: the GENES study[END_REF], the study's participants (n = 99) were stable CHD patients, ages 45-74, living in the Toulouse area (southwest France) and prospectively recruited from 2001 to 2004 after admission to the Cardiology Department, Toulouse University Hospital, for cardiovascular examination and referred for evaluation and management of their CHD. During the same period, individuals of the same age range as the study participants but without CHD (further referred to as controls, n = 105) were selected from the general population using electoral rolls. Pools of human EDTA plasma and serum (8 donors; 50% males) were also purchased from the French Blood Bank (Nantes, France).

Standard samples and quality controls

Quality control (QC) samples were prepared at 4 concentration levels (0, 100, 600, 1500 ng/mL; i.e. 0, 10, 63, 158 nM). Concentrated solutions of human IF1 protein were prepared in water and then diluted 20-fold in pools of plasma (QC samples) or in water (control samples). In parallel, a mixed solution of unlabelled proteotypic peptides was constituted and serially diluted in water to obtain seven standard solutions, ranging from 200 to 10 nM (i.e. equivalent to 1,903-95 ng/mL of human IF1). A mixed solution of labelled peptides (10 µM) was prepared and added to the digestion buffer to create a final concentration of 100 nM. QC samples served both to control the repeatability and accuracy of the assay and to ensure that the enzymatic proteolysis is optimal as we used synthetic proteotypic peptides for calibration.

Sample preparation

Samples were prepared with the ProteinWorks TM eXpress kit using an optimized procedure. Samples (40 µL) were incubated for 10 min at 80 °C in digestion buffer containing labelled peptides (100 µL) and RapidGest detergent solution (7 mg/mL, 10 µL), reduced for 20 min at 60 °C with dithiothreitol (20 µL), alkylated for 30 min at room temperature in the dark with iodoacetamide (30 µL), and digested for 8 hours at 37 °C with trypsin (30 µL). Enzymatic digestion was stopped with 20% TFA (5 µL). After 15 min at 45°C, precipitates were removed by centrifugation (15 min, 10 °C, 10,000 × g), and supernatants were cleaned using 30-mg Oasis HLB Cartridges (Waters Corporation), which were conditioned (100% MeOH; 1 mL), equilibrated (100% water; 1 mL), loaded (sample; ~200 µL), washed (5% MeOH, 0.1% TFA; 1 mL), and eluted (60% MeOH, 0.1% TFA; 500 µL). Eluates were dried under a gentle stream of nitrogen (45 °C), reconstituted with 10% AcN containing 0.1% FA (70 µL), and injected into the LC-MS/MS system.

Analytical parameters

Analyses were performed on a Xevo ® TQD mass spectrometer with an electrospray ionization (ESI) interface and an Acquity H-Class ® UPLC TM device (Waters Corporation). Labelled and unlabelled peptides were separated on an Acquity ® BEH C18 column (1.7 µm. 2.1 × 100 mm, Waters Corporation) held at 60 °C with a linear gradient of mobile phase B (AcN containing 0.1% FA) in mobile phase A (5% AcN in water containing 0.1% FA) and at a flow rate of 500 µL/min. Mobile phase B was linearly increased from 1% to 70% for 5 min, kept constant for 1 min, returned to the initial condition over 1 min, and kept constant again for 1 min before the next injection. Subsequently, reconstituted sample (10 µL) were injected into the LC column. Labelled and unlabelled peptides were then detected by the mass spectrometer with the ESI interface operating in the positive ion mode [capillary voltage, 3 kV; desolvation gas (N2) flow and temperature, 1000 L/h and 450 °C; source temperature, 120 °C]. The multiple reaction monitoring (MRM) mode was applied for MS/MS detection. Selected MRM transitions, cone voltages, and collision energies (Table 1) were optimized by the IntelliStart interface (Waters Corporation) and the infusion of individual peptide stock solutions (20 µM, MeOH). Data acquisition and analyses were performed using MassLynx® and TargetLynx® software, respectively (version 4.1, Waters Corporation). Dwell times were set at 128 ms for all MRM transitions with a quadrupole filtering mass window set at ±0.2 Da. The method was forced to get at least 15 points per peaks for all targeted MRM transitions (peak widths = 5 s). Peak integration was performed with the following parameters: smoothing method "Mean"; smoothing iteration = 1; smoothing width = 1; all standards peak detection parameters = 0; and threshold parameters = 1.5 (relative height and area).

Optimization of sample preparation

The method was developed and optimized using QC samples spiked with known amounts of human IF1 protein. For trypsin proteolysis, 8 incubation times (0, 1, 2, 3, 4, 8, 16 and 24 hours) were assessed (n = 6). Solid-phase extraction (SPE) was also optimized from QC samples treated as described above. The wash and elution conditions were optimized with increasing levels of MeOH or AcN in water, with or without additives (0.1% TFA or 0.5% NH4OH), added directly after sample loading (n = 3). Several mixtures of AcN in water containing 0.1% FA were also tested to reconstitute sample after drying of peptide solutions at 50 nM (n = 3).

Method validation

The LC-MS/MS assay was validated via four independent experiments with two calibration curves and 24 plasma QCs (4 concentration levels; n = 6). The assay linearity was illustrated by R² coefficients calculated from calibration curves by linear regression analysis (1/x weighting, origin excluded). The intra-and inter-assay variability was expressed by coefficients of variation (CVs) obtained at each QC level. The assay accuracy was expressed by the mean bias between the theoretical and measured concentrations at each QC level after baseline subtraction (i.e., the mean measured concentration of unspiked QC). The targeted lower limit of quantification (i.e. 100 ng/mL of IF1) was validated with a signal-to-noise ratio greater than 10 and a variability and accuracy lower than 20%. Limits of acceptance were set at ± 15% for accuracy and variability at other concentration levels [START_REF] Allinson | Clinical biomarker validation[END_REF]. Crossvalidation was performed by comparing results obtained by standard ELISA and LC-MS/MS on plasma samples from 204 subjects. For quantitative experiments, plasma samples spiked with 375 ng/ml (5 nM) of human IF1 (n = 3) or digestion buffer (n = 3) were used to check the completion of the enzymatic reaction and the reliability of the assay.

Data management and statistical analysis

IF1 protein concentrations were calculated using calibration curves plotted from standard solutions.

One mole of peptide was assumed to be equivalent to 1 mole of peptide was equivalent to 1 mole of protein. The concentrations were then converted to standard units (ng/mL) using the IF1 molecular weight (9,517 g/mol). Data are presented as percentages for qualitative variables and as means with standard deviations (SD) for quantitative ones. The Shapiro-Wilks test was used to test the normality of distribution of residuals. A Spearman correlation test and a Bland-Altman plot were generated to compare both methods [START_REF] Bland | Statistical methods for assessing agreement between two methods of clinical measurement[END_REF]. For the Bland-Altman plot, the difference of values (y-axis) obtained from the two methods was plotted according to the average value obtained with the two methods. The mean difference and the limits of agreement, corresponding to the 95% confidence level (i.e., mean ± 1.96 × SD), were calculated. The degree of correlation and agreement between measurements was assessed by calculating the intra-class correlation coefficient (ICC) [START_REF] Shrout | Intraclass correlations: Uses in assessing rater reliability[END_REF]. Comparison between groups was made using two-tailed unpaired Student t-test. Spearman coefficients were calculated to study the correlations between IF1 and HDL-C. Outcomes of p <0.05 were considered as statistically significant.

Graphics and statistical analyses were achieved with GraphPad Prism software (version 6.0, GraphPad Software Inc., La Jolla, CA, USA) and STATA software (release 14.1, Stata Corporation, College Station, TX, USA).

Results and Discussion

Selection of peptide markers

human mature IF1 protein. Peptides and their labelled internal standards were thus synthesized for optimization of the MRM transitions used for LC-MS/MS analyses (Table 1). Candidate peptides were primarily detected as doubly charged precursor ions. After MS/MS fragmentation, each precursor ion yielded several specific and singly charged "y" (C-term) or "b" (N-term) product ions, thereby ascertaining the peptide sequences. For each candidate peptide, the most intense product ion was selected to quantify human IF1 protein levels. Moreover, the product ion second in intensity was kept to confirm the specific detection of the target peptide in biological samples. Noteworthy, the presence of one glutamic acid on peptide #3 and one glutamic acid and one glutamine on peptide #4 could lead the formation of pyroglutamate formation by ionization-induced or gas-phase reactions. Such reaction was monitored by the selection of specific MRM transitions (i.e. pyro_EAGGAFGK: m/z 359.8 → 518.4 and pyro_EQLAALK: m/z 377.9 → 129.1). Whereas the pyroglutamic acid formation was shown negligible for peptide #3 (~0.4%), this reaction was found more pronounced for peptide #5 (~5%).

Optimization of sample preparation

Optimization of trypsin proteolysis showed contrasting released profiles between candidate peptides (Fig. 1A). Contrary to manufacturer's recommendations (2h, 45 °C), the optimal condition was reached with a trypsin incubation at 37 °C for 8 h. However, only two peptides were satisfactorily generated (#3: EAGGAFGK; #5: EQLAALK). Our further investigations revealed that peptide #1, peptide #2, peptide #4 and peptide #6 displayed several deleterious properties, including a more pronounced matrix effect, a poor stability during sample preparation, and a poor ionization yield, as compared to peptide #3 and peptide #5. Hence, these peptides were not considered for the assay validation. All other manufacturer's recommendations (linearization, reduction, and alkylation) were confirmed by testing four incubation times on the pooled human plasma (not shown). Moreover, SPE was also optimized on plasma samples spiked with known amounts of recombinant IF1 (QC).

Moreover, MeOH was slightly more efficient than AcN for peptide recovery, and an acidic additive (TFA) markedly improved extraction by up to 40%. The optimized wash and elution solvents used were 5% and 60% methanol containing 0.1% TFA, respectively (Fig. 1B). After drying, several mixtures were tested for sample reconstitution to obtain the optimal signal intensity. The optimal mixture was determined to be 10% AcN containing 0.1% TFA for peptide #3 and peptide #5 (Fig. 1C).

Higher concentrations of AcN led to peak distortions and signal loss. Despite SPE, plasma strongly reduced peptide detection by ~25%, compared with aqueous controls. This could be related to contaminations coming from high abundance proteins or other interferences since we did not use prepurification methods such as immuno-capture or -depletion prior proteolysis. However, except for peptide #2, all of the peptide candidates were highly specific of human IF1 and did not cross with other human protein sequences. These matrix effects were corrected after internal standard normalization.

Of note, serum was also tested and showed higher signal loss (~60%) with a more pronounced variability than that observed with plasma. Different plasma volumes [START_REF] Bland | Statistical methods for assessing agreement between two methods of clinical measurement[END_REF]20,40, and 50 µL) were also assessed and 40 µL was found to be the most suitable for assay sensitivity and proteolysis efficiency (not shown).

Method validation

To test the linearity of the method, a regression model (1/x weighting and origin excluded) was used.

After analysis of the 8 calibration curves (2 calibration curves per experiment), the mean R² value were 0.998 ± 0.002 and 0.996 ± 0.001 for peptide #3 and peptide #5, respectively, and the CVs never exceeded 9.1% over the concentration range tested (Table 2). Of note, at the targeted lower limit of quantification (95 ng/mL of human IF1), the signal-to-noise ratios were of 23 ± 4 and 7 ± 2 for peptide #3 and peptide #5, respectively. Human IF1 was also found to remain stable for 6 months at -80 °C and after 3 freeze/thaw cycles (-80 °C) in human EDTA plasma. Stock solutions of the synthetic peptides #3 and #5 were also stable for 6 months at -20 °C. Finally, digested samples were found stable for 5 days in the refrigerated autosampler (10 °C) and 6 months at -20 °C (not shown). Intraand inter-assay variability parameters were then assessed using calibration curves and QC samples.

For peptide #3, intra-and inter-assay CVs did not exceed 14.2% and the mean measured concentrations did not deviate by more than 4.9% (mean bias) as compared to expected concentrations (Table 2). In contrast, intra-and inter-assay CVs were out of our acceptance criteria for peptide #5 (± 15%) [START_REF] Croyal | Multiplexed peptide analysis for kinetic measurements of major human apolipoproteins by LC/MS/MS[END_REF][START_REF] Allinson | Clinical biomarker validation[END_REF], likely as a result of the pyroglutamic acid formation occurring for this peptide.

These results indicate that solely peptide #3 (EAGGAFGK) can be used to accurately quantify human IF1 in plasma samples, and was thus retained for further analyses. Its specificity was further investigated in bovine plasma that we used as a surrogate matrix as bovine IF1 sequence is distinct than that of human. At the targeted lower limit of quantification, we confirmed the absence of interfering peaks (blank plasma) and a signal-to-noise ratio greater than 20 in bovine plasma spiked with human IF1 protein (100 ng/mL).

Comparison of LC-MS/MS with ELISA

For method comparison, we used 204 human plasma samples from the GENES study, in which IF1 levels were previously determined by the ELISA assay [START_REF] Genoux | Serum IF1 concentration is independently associated to HDL levels and to coronary heart disease: the GENES study[END_REF]. As shown in Fig. 2A, IF1 concentrations measured by LC-MS/MS from peptide #3 (n = 204) were significantly correlated with those obtained by ELISA (r =0.89, p <0.001). Intraclass correlation coefficient was 0.93 [95% CI, 0.91-0.95], indicating an excellent degree of correlation and agreement between LC-MS/MS and ELISA measurement. In the Bland-Altman test, the mean of both concentrations obtained by LC-MS/MS and ELISA was calculated and plotted against the difference between both measurements. The mean difference and limits of similarity, corresponding to the 95% confidence interval, were drawn and it was demonstrated that 195 out of the 204 values were within these limits (Fig. 2B). However, the Bland-Altman plot highlighted non-negligible concentration differences between the analytical approaches, with deviations of up to 50%, but only 9 outliers were identified (< 5%). After exclusion of these outliers, the mean deviation between both approaches was 3.4 ± 13.7 %. Hence, the human IF1 protein concentrations measured by LC-MS/MS were considered similar to those obtained by ELISA.

Clinical validation

One of the main features of circulating IF1 is that its levels, measured by ELISA assay, were positively correlated with levels of HDL-C (r = 0.40, p< 0.001) [START_REF] Genoux | Serum IF1 concentration is independently associated to HDL levels and to coronary heart disease: the GENES study[END_REF]. Accordingly, from the Spearman correlations analysis, plasma IF1 measured by LC-MS/MS with peptide #3 was found to positively correlate with HDL-C (r = 0.38, p< 0.0001, Fig. 2C). Another important clinical output of measuring circulating IF1 level is that it was found to be significantly lower in CHD patients as compared to control individuals [START_REF] Genoux | Serum IF1 concentration is independently associated to HDL levels and to coronary heart disease: the GENES study[END_REF]. In the present cohort, the mean IF1 concentrations were found significantly lower in CHD patients than in controls when measured by LC-MS/MS (431 ± 132 ng/mL and 555 ± 173 ng/mL, p <0.0001) and ELISA (431 ± 118 ng/mL and 558 ± 180 ng/mL, p <0.0001). Altogether, these results confirmed that the LC-MS/MS method we have developed as part of the present study is clinically relevant for the accurate quantification of plasma IF1 levels in human samples.

Towards IF1 isoforms and polymorphisms

In humans, IF1 is a nuclear encoded protein translated as a precursor protein that undergoes the removal of the N-terminal 25-residue pre-sequence when imported to the mitochondria. At this stage, it is still unknown whether IF1 is circulating as the precursor or mature form. Our study does not allow to discriminate these forms since all selected peptide markers were present in the mature sequence of IF1. This question could be further investigated by targeting additional proteotypic peptides located into the signal peptide sequence of the protein (MAVTALAAR, TWLGVWGVR and TMQAR). However, due to the low plasma concentrations of human IF1, such determination could require specific enrichment of the protein prior mass spectrometry analysis (e.g. immuno-precipitation with polyclonal antibodies). Three IF1 isoforms, resulting from alternative splicing at ATPIF1 gene locus, have been described in humans [START_REF] Genoux | Mitochondrial Inhibitory Factor 1 (IF1) Is Present in Human Serum and Is Positively Correlated with HDL-Cholesterol[END_REF]. IF1 variant 1 (NCBI Reference Sequence: NP_057395.1) encodes the longest isoform, while IF1 variant 2 (NCBI Reference Sequence: NP_835497.1) and variant 3 (NCBI Reference Sequence: NP_835498.1) have a shorter and distinct C-terminus compared to isoform 1. The selected peptide #3 used in the present LC-MS/MS method is located within a sequence common to the three variants and thus enables the quantification of the 3 isoforms together. Among the candidate peptide markers (Table 1), peptides #5 and #6 are specific of isoform 1. However, peptide #6 was not efficiently released after trypsin proteolysis and validation parameters for peptide #5 were not within our acceptance criteria (Table 2). Hence, further studies in developing a methodology using one of these peptides will be warranted to specifically quantify isoform 1. However, a recent study performed in several human tissues reported negligible mRNA expression for isoforms 2 and 3 compared to isoform 1 [START_REF] Esparza-Moltó | Tissue-specific expression and post-transcriptional regulation of the ATPase inhibitory factor 1 (IF1) in human and mouse tissues[END_REF], suggesting that isoform 1 is likely to be the major circulating isoform. Additionally, previous studies demonstrating the clinical interest of IF1 measurement in the circulation have been performed using polyclonal antibodies in competitive ELISA assays that did not differentiate between various IF1 isoforms [START_REF] Genoux | Mitochondrial Inhibitory Factor 1 (IF1) Is Present in Human Serum and Is Positively Correlated with HDL-Cholesterol[END_REF][START_REF] Lee | ATP synthase inhibitory factor 1 (IF1), a novel myokine, regulates glucose metabolism by AMPK and Akt dual pathways[END_REF], thus indicating that the clinical significance of IF1 is measurable with a method that does not specifically quantify isoform 1.

Regarding the current knowledge on the potential clinical impact of IF1 polymorphisms, we found that the single nucleotide polymorphism rs748968212 (Highest population Minor Allele Frequency (MAF) < 0.01%, Ser39 > Phe39) could affect the phosphorylation state of IF1 and modulate its inhibitory activity on ATP synthase [START_REF] García-Bermúdez | PKA Phosphorylates the ATPase Inhibitory Factor 1 and Inactivates Its Capacity to Bind and Inhibit the Mitochondrial H+-ATP Synthase[END_REF]. Interestingly Ser39 is located in the candidate peptide marker #2 (Table 1). In the future, developing a methodology using this peptide might serve to quantify circulating levels of IF1 in heterozygous patients for this mutation, as it has been previously developed for other isoforms of circulating proteins, such as apoE, for which production or catabolic rates are differently affected according to the specific isoforms [START_REF] Blanchard | Kinetics of plasma apolipoprotein E isoforms by LC-MS/MS: a pilot study[END_REF].

Conclusion

Here, we have developed and validated a simple and fast LC-MS/MS assay enabling the accurate quantification of human IF1 in plasma samples into the range 100-1,500 ng/mL (i.e. 10-160 nM).

Unlike most conventional LC-MS/MS based assays for low-abundancy proteins, protein concentration by immunoaffinity or immunodepletion was not necessary for our assay. Of note, LC-MS/MS allows a high level of multiplexing, enabling simultaneous quantification of several proteins and providing further information on a patient's metabolic profile. In that respect, we have already described a validated LC-MS/MS assay for the quantification of several apolipoproteins in a single run [START_REF] Croyal | Multiplexed peptide analysis for kinetic measurements of major human apolipoproteins by LC/MS/MS[END_REF][START_REF] Croyal | Fenofibrate decreases plasma ceramide in type 2 diabetes patients: A novel marker of CVD?[END_REF]. Here, the proposed method was multiplexed for the simultaneous analysis of IF1 and major HDL-related apolipoproteins such as apoA-I, apoA-II, apoE, apoD and apoM ( [START_REF] Croyal | Multiplexed peptide analysis for kinetic measurements of major human apolipoproteins by LC/MS/MS[END_REF] and data not shown), thus making it amenable for a wide range of studies aiming to decipher the role of IF1 in CHD. Finally, the LC-MS/MS method developed in our study could be used in the future to determine IF1 turnover in vivo by measuring the incorporation of an injected labelled tracer, such as 2 H3-leucine, over time, thus allowing the determination of its kinetic parameters, including production and catabolic rates [START_REF] Blanchard | Kinetics of plasma apolipoprotein E isoforms by LC-MS/MS: a pilot study[END_REF].

Acknowledgements

We are grateful to the Biogenouest Corsaire core facility for their financial support.

Funding

This work was supported by the French National Research Agency (ANR, #ANR-16-CE18-0014-01), European Regional Development Fund (ERDF, Fonds Européen pour le Développement Régional) and "La Région Occitanie" (Project THERANOVASC n° ESR_R&S_DF-000094/2018-003303/18009464, Project HEPATOCARE n°19014226).

Authorship contribution statement

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The analytical validation was performed over four distinct experiments. Assay linearity was illustrated by the R² coefficients and slopes (mean ± SD, n = 8) calculated from calibration curves by linear regression analysis (origin excluded, 1/x weighting). Intra-and inter-assay variability was expressed by the coefficients of variation (CVs) obtained at each QC level (n = 6 × 4 per level). Assay accuracy was expressed by the mean bias between the theoretical and measured concentrations after baseline subtraction (n = 6 × 4 per level). Stabilities were evaluated on QC samples stored at -80 °C and assayed with fresh calibration curves (n = 6 × 4). N/A: not applicable.

Figure legends

Graphical abstract (color should be used)

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