Celine Chollet

Stéphanie Boutet-Mercey

Laurent Laboureur

Carlos Rincon

Marie Méjean

Juliette Jouhet

Francois Fenaille

Benoit A Colsch

David Touboul
email: david.touboul@cnrs.fr

Céline Chollet

Laboureur Laurent

François Fenaille

Supercritical fluid chromatography coupled to mass spectrometry for lipidomics

come

Introduction

Lipids can be defined as a set of hydrophobic biomolecules with extremely varied physicochemical structures and properties that play multiple biological functions in living organisms.

They are an important energy source for organisms and accomplish essential functions in cell compartmentalization and signaling. [START_REF] Spector | Membrane lipid composition and cellular function[END_REF] However, all of their functions are still not fully known and understood. In human health, lipids are scrutinized for the discovery of biomarkers of pathologies. Indeed, alterations or variations in the concentration of lipids are indicative of dysfunctions in the body that may be linked to pathologies, such as cancer, autoimmune diseases, diabetes and inherited metabolic diseases. [START_REF] Stephenson | Lipidomics in translational research and the clinical significance of lipid-based biomarkers[END_REF][START_REF] Lamari | Disorders of phospholipids, sphingolipids and fatty acids biosynthesis: toward a new category of inherited metabolic diseases[END_REF] In the field of renewable energies, the ability of certain algae to accumulate high levels of some particular lipid species can be exploited for the production of biofuels. [START_REF] Khan | Omics Technologies for Microalgae-based Fuels and Chemicals: Challenges and Opportunities[END_REF] Over the last two decades, technical progress in the development of analytical methods, combined with the growing interest in lipids, has led to a broad development of lipidomic studies. [START_REF] Gross | The evolution of lipidomics through space and time[END_REF][START_REF] Han | Lipidomics for precision medicine and metabolism: A personal view[END_REF] The great diversity of lipids in terms of structure and physico-chemical properties as well as a very wide concentration range (pg/g to mg/g of dried tissue) make it difficult to simultaneously separate, detect and annotate all lipid signals in complex biological matrices.

The most common chromatographic technique used for lipid analysis is high performance liquid chromatography (HPLC) in normal phase (NPLC) or reverse phase (RPLC). In RPLC, lipids are separated by molecular species according to their polarity depending on the polar head, carbon chain lengths, the number of unsaturations and the presence of hydroxylations and methylations. This method leads to the elution of many lipids of different classes in a reduced chromatographic space that can induce significant ion suppression phenomena. In addition, this chromatographic mode is not compatible with the injection of organic solvents such as chloroform or hexane requiring an extra sample preparation step (drying and resuspension in alcohol). In the opposite, NPLC allows separation by lipid class and is compatible with organic solvent for injection. Nevertheless, the analysis is time consuming (about 30 minutes) and require the use of toxic organic eluents. Gas chromatography (GC) remains mainly focused on fatty acid analysis after saponification of complex lipids and methylation of free fatty acids.

The recent improvement of chromatographic technology based on the use of subcritical/supercritical fluids is of increasing interest to lipidomics research groups. [START_REF] Laboureur | Lipidomics by Supercritical Fluid Chromatography[END_REF] Indeed, supercritical fluid chromatography (SFC) is considered a hybrid of GC and LC, and has many advantages, such as high separation efficiency, low organic solvent consumption and short analysis time, up to 4 times when comparing LC and SFC with the same column parameters (internal diameter, length and particle size), required for high-throughput screening. [START_REF] Zhang | Supercritical fluid chromatography-tandem mass spectrometry for high throughput bioanalysis of small molecules in drug discovery[END_REF] It is particularly well suited for the analysis of low-polarity compounds, such as lipids, because supercritical CO2 is an non-polar solvent, often compared to hexane. [START_REF] Lesellier | The many faces of packed column supercritical fluid chromatography--a critical review[END_REF] With some adaptations, which will be described hereafter, SFC can be coupled with mass spectrometry (MS) techniques, providing high mass resolution, mass accuracy and tandem MS in particular, to allow efficient separation and annotation of complex lipid mixtures.

History and principles of SFC

Nowadays, carbon dioxide (CO2) is by far the most widely used supercritical fluid as a mobile phase in SFC. It is the only fluid for which commercial chromatography systems are available today. The discovery of CO2 in the supercritical phase dates back to 1822 by Baron Charles Cagniard de la Tour (1777-1859), who introduced his work to the "Académie des Sciences" in 1823. [START_REF] Ampère | Rapport fait à l'Institut de France le 27 octobre 1823 sur un mémoire de M. Cagniard de La Tour[END_REF] Andrews first described this state as "critical" in 1869 when he determined the critical pressure and temperature (31°C and 74 bar) of CO2. 11 These two values are fully compatible with pumps and columns conventionally used in LC. Supercritical CO2 displays the advantage of not being toxic to humans at low concentrations, of being chemically inert towards most chemical compounds, non-flammable, odourless and inexpensive. It is also UV transparent as low as 190 nm, allowing the hyphenation with UV detectors using high-pressure cells. The only limitation could be its low solvation power, similar to hexane. [START_REF] Nováková | Modern analytical supercritical fluid chromatography using columns packed with sub-2 μm particles: a tutorial[END_REF] Nevertheless, this limitation is mitigated by the miscibility of supercritical CO2 with the majority of organic solvents (alcohols, acetonitrile). It is therefore possible to modify its solvation power by forming binary CO2/solvent mixtures. The first chromatographic separation using a supercritical fluid as a mobile phase is attributed to Klesper et al. in 1962. [START_REF] Klesper | High Pressure Gas Chromatography above Critical Temperatures[END_REF] The mobile phase then consisted of a halogenated alkane and the stationary phase of polyethylene glycol.

In its early days, SFC was considered as an extension of gas chromatography (GC). The authors describe it as a chromatography using a dense gas phase allowing the study of thermolabile compounds. The use of a restrictor provided the necessary pressures in the chromatographic columns. Since then, two types of chromatographic columns coexisted until the late 1990s: capillary and packed columns.

The capillary SFC (cSFC) used capillary or "open tubular" columns derived from GC columns. [START_REF] Peaden | Instrumental aspects of capillary supercritical fluid chromatography Anal[END_REF] They consisted of a fused silica capillary with an internal diameter of 50 μm and a stationary phase film on the wall. High pressure was maintained using a restrictor at the outlet of the chromatographic column. The instrumentation was similar to that used in GC, notably for detection using a flame ionization detector (FID). The FID was perfectly suited when using pure CO2 since it does not provide a FID signal. However, FID is incompatible with mobile phases consisting of a binary mixture (CO2/polar modifier) since the polar modifier leads to a high FID signal. Until the early 1990s, cSFC was the most widespread SFC technique. The use of CO2 at low flow rates (a few mL/min) also allowed coupling to mass spectrometry. However, cSFC shows long analysis times and low column efficiencies compared to GC with the same column. [START_REF] Morrissey | Ion-mobility spectrometry as a detection method for packedcolumn supercritical fluid chromatography[END_REF] This type of column is now very uncommon in SFC since all commercial systems use packed columns.

The packed-SFC consists of the use of packed columns and reflects the fact that the SFC can also be considered as the extension of liquid chromatography. In 1983, Hewlett-Packard launched the first commercial SFC system. This system is directly based on a commercial HPLC system. The use of packed columns increases the interaction surfaces with the stationary phase and reduces dead volumes. [START_REF] Taylor | Supercritical fluid chromatography for the 21st century[END_REF] Due to the low viscosity of supercritical fluids (Table 1), the pressure drops in the system are lower than in LC. It is therefore technically possible to increase the flow rate of the mobile phase without the risk of reaching the instrumental limits. Moreover, considering the high diffusion coefficient value, this increase in flow rate has no impact on the quality of the separations. These properties often lead to flow rates that are 3 times higher in SFC than LC, but at the same time, a lower organic solvent consumption since most of the mobile phase is composed of CO2. As in LC, the mobile phase can be made up of a binary mixture: a co-solvent (or polar modifier) is added to the CO2 in order to modulate the elution strength of the mobile phase. [START_REF] Crowther | Supercritical fluid chromatography of polar drugs using smallparticle packed columns with mass spectrometric detection[END_REF] In practice, the most commonly encountered co-solvent is ethanol since it allows a high modulation of elution strength, is available in analytical grade, is inexpensive and bears a relative low toxicity. Other solvents such as methanol, isopropanol and less frequently acetonitrile are also used. This strategy allows the study of more polar compounds and extends the scope of the SFC, particularly to pharmaceutical compounds. [START_REF] Pinkston | Advantages and drawbacks of popular supercritical fluid chromatography / mass spectrometry interfacing approaches -a user's perspective[END_REF] However, when the compounds studied are polar, the mobile phase composed of a binary mixture no longer reaches satisfactory chromatographic performance. The obtained peaks are then extremely deformed. Figure 1 shows an example from which the addition of ammonium acetate at a concentration of 15 mmol/L in the co-solvent significantly improved peak shape. The most common additives are acetic and formic acids, ammonium acetate or formate, and water. It should be noticed here that water is considered as an additive and not a co-solvent. Indeed, since water and CO2 are not miscible, only a few percent (1 to 5 %) of water can be added to the co-solvent. [START_REF] West | Characterization and use of hydrophilic interaction liquid chromatography type stationary phases in supercritical fluid chromatography[END_REF] Additives can act in different ways. For example, they can avoid ionization (by modifying the pH), interact with active sites of the stationary phase or form ion pairs with charged analytes. [START_REF] Berger | Chromatography: supercritical fluid, theory of supercritical fluid chromatography[END_REF] These additives can sometimes significantly modify the stationary phase and thus modify the characteristics of a column. When coupling to mass spectrometry, the additive must be carefully chosen not to interfere with the ionization of analytes or pollute the analyzers. Amines (isopropylamine, dimethylamine, triethylamine), quaternary amine salts and trifluoroacetic acid should be thus avoided.

Given the compressibility of the mobile phase, a back pressure regulator (BPR) is required to maintain the mobile phase pressurized through the system. This regulator is an automated valve that imposes pressure at the outlet of the column regardless of the flow rate through it.

Although very important when pure CO2 is used as a mobile phase, the value of the back pressure generally shows a weak influence when a binary mobile phase is used. Effects on retention are minimal and those on selectivity are almost non-existent. To maximize the chances of observing effects, it is necessary to make changes with relatively large steps (at least 20 bar).

There is no ideal and universal stationary phase chemistry in SFC. However, the choice of the stationary phase can be based on the work of Lesellier's group. [START_REF] Lesellier | Overview of the retention in subcritical fluid chromatography with varied polarity stationary phases[END_REF] They propose a classification by studying the retention factors (k) obtained when separating a mixture of 109 molecular compounds under identical chromatographic conditions (except the stationary phase chemistry). This classification is based on column characterization via quantitative structureretention relationships (QSRRs).

The equation to be solved for each column is as follows:

𝑙𝑜𝑔𝑘=𝑐C+𝑒𝐸+𝑠𝑆+𝑎𝐴+𝑏𝐵+𝑣𝑉+𝑑 -𝐷 -+𝑑 + 𝐷 +
Upper-case letters are variables corresponding to each compound. Lower-case letters are the constants to be determined for each column. c is the intercept term for the model. E is linked to the refractive index of the analyte, S to the polarity/apolarity of the analyte, A and B to the acid and basic characteristics of the hydrogen bonds, V is the McGowan volume, D + represents the charges carried by a cation and D -those carried by an anion. [START_REF] West | An improved classification of stationary phases for ultra-high performance supercritical fluid chromatography[END_REF] Initially the D terms were not included in the original model. [START_REF] West | A unified classification of stationary phases for packed column supercritical fluid chromatography[END_REF] This resulted in poor classification when ionic or zwitterionic species were considered. However, the calculation of these constants causes problems since it requires knowledge of both pH and pKa in situ.

Indeed, the pKa of an analyte depends on its environment and is not measurable in SFC.

Similarly, the pH of the mobile phase is difficult to measure experimentally. West et al. propose the use of colored indicators and estimate the "apparent aqueous" pH of the mobile phase CO2/MeOH (with or without basic additives) to be between 2 and 8 and close to 1 when using acid additives. [START_REF] West | Unravelling the effects of mobile phase additives in supercritical fluid chromatography. Part I: Polarity and acidity of the mobile phase[END_REF] A double approximation was therefore agreed by the authors: use a pH value equal to 5 [START_REF] Wen | Characterization of pH in liquid mixtures of methanol/H2O/CO2[END_REF] and the pKa obtained in aqueous solution. The final results are presented in the form of a spider diagram where each axis corresponds to a constant (Figure 2). Although very useful to get an idea of columns with similar or orthogonal selectivities, this classification can show some limitations, especially when using additives or a co-solvent significantly different from an alcohol. [START_REF] Snyder | Classification of the solvent properties of common liquids[END_REF] For these reasons, each new project starts with a column screening in our laboratory.

Currently, the range of commercial equipments is quite limited. Indeed, almost all authors in the literature use UPC² system (Waters) or Infinity 1260 SFC system (Agilent Technologies).

It should be noted that other manufacturers sell analytical SFC systems (Shimadzu, Pic Solution or Jasco). All these systems use chromatographic columns with geometries similar to liquid chromatography (Figure 3). These systems are based on the knowledge and experience of ultra-high performance liquid chromatography (UHPLC) manufacturers since they operate at pressures comparable to SFC. The commercialization of these reliable and robust systems has enabled SFC to find its place alongside LC and GC in the world of analytical chemistry.

SFC-MS hyphenation

Mass spectrometry is the most widely used detection system for lipidomics studies. The coupling of SFC with MS is highly valuable because it combines the efficiency and speed of the SFC separation with the contribution of the high sensitivity and specificity of MS detection.

Atmospheric pressure sources such as electrospray (ESI), atmospheric pressure photoionization (APPI) and atmospheric pressure chemical ionization (APCI) are well suited for coupling with SFC and allow the analysis of compounds with different polarities. [START_REF] Méjean | Quantification of tocopherols and tocotrienols in soybean oil by supercritical-fluid chromatography coupled to high-resolution mass spectrometry[END_REF][START_REF] Laboureur | Structural Characterisation of Acetogenins from Annona muricata by Supercritical Fluid Chromatography Coupled to High-Resolution Tandem Mass Spectrometry[END_REF][START_REF] Laboureur | Profiling of modified nucleosides from ribonucleic acid digestion by supercritical fluid chromatography coupled to high resolution mass spectrometry[END_REF][START_REF] Santerre | Fingerprints of flower absolutes using supercritical fluid chromatography hyphenated with high resolution mass spectrometry[END_REF] All mass analyzers can be coupled to the SFC, and the choice will depend on the study issue: low-resolution analyzers, such as the quadrupole, only provide access to the nominal mass of the compound, while high-resolution analyzers, such as Orbitrap or Time-of-Flight (TOF) analyzers, provide access to the accurate mass of the ions. In the case of studies of complex biological environments, the use of high-resolution mass spectrometry (HRMS) is most often necessary for untargeted profiling whereas triple quadrupoles are required for targeted quantitative approaches.

The coupling of atmospheric pressure ionization sources with SFC is significantly more challenging than with LC. Although the properties of the supercritical fluid are favorable for nebulization, its expansion at the outlet of the column results in poor solvation of the solutes.

The latter may then precipitate into the transfer capillary to the ionization source. To avoid this problem and facilitate the ionization of the compounds, a solvent, called a "make-up" solvent, is added using an isocratic pump and a "T" or "Y" type fitting. The choice of this solvent depends on the nature of the compounds to be analyzed and must be optimized in order to increase the solubility of the components and promote their ionization. For this purpose, it is possible to test any type of solvent and additives, ensuring that they are miscible with the mobile phase and compatible with the ionization source. The addition of a make-up solvent is required for lipid quantifications for better repeatability. Finally, in order to avoid the condensation of CO2 during its expansion, a module called "caloratherm", consisting of a heated sheath whose temperature is regulated to a chosen value, is placed just before the source.

Several types of interfaces were reviewed and compared by Pinkston first in 2005, [START_REF] Pinkston | Advantages and drawbacks of popular supercritical fluid chromatography / mass spectrometry interfacing approaches -a user's perspective[END_REF] then by Grand-Guillaume Perrenoud et al. in 2014. [START_REF] Grand-Guillaume Perrenoud | Coupling state-of-the-art supercritical fluid chromatography and mass spectrometry : From hyphenation interface optimization to high-sensitivity analysis of pharmaceutical compounds[END_REF] Very recently, Tarafder published a review of the different solutions proposed for coupling SFC to MS (Figure 4), and their effects on dispersion and chromatographic resolution. [START_REF] Tarafder | Designs and methods for interfacing SFC with MS[END_REF] Two configurations are currently operated for coupling with a mass spectrometer: either the coupling takes place after the back pressure regulator BPR, and in this case all the chromatographic effluent is injected into the ionization source, or it can be done before the BPR, using a split system. Although the first configuration (Figure 4d) allows to obtain a good sensitivity, BPR generates important dead volumes which induce a consequent loss of chromatographic efficiency. The introduction of the split system makes it possible to minimize dead volumes (Figure 4e-f). Even if only a part of the sample is introduced, the effects of dilution would be compensated by thinner chromatographic peaks. In addition, the possibility of adding make-up solvent after BPR has also been described in the literature.

Few systematic studies have compared the ionization efficiency of ESI, APCI and APPI sources in coupling with SFC. Méjean et al. reported that the APPI source is significantly more sensitive and robust than ESI and APCI for the quantification of tocopherols and tocotrienols in edible oils. [START_REF] Méjean | Quantification of tocopherols and tocotrienols in soybean oil by supercritical-fluid chromatography coupled to high-resolution mass spectrometry[END_REF] To obtain the best ionization conditions, all voltage and gas pressure parameters must be carefully optimized. In this sense, Duval et al. published an extensive study on the influence of different source parameters in APCI for the detection and quantification of lipids in vegetable oils. [START_REF] Duval | Hyphenation of ultra high performance supercritical fluid chromatography with atmospheric pressure chemical ionisation high resolution mass spectrometry: Part 1. Study of the coupling parameters for the analysis of natural non-polar compounds[END_REF][START_REF] Duval | Hyphenation of ultra-high performance supercritical fluid chromatography with atmospheric pressure chemical ionisation high resolution mass spectrometry: Part 2. Study of chromatographic and mass spectrometry parameters for the analysis of natural non-polar compounds[END_REF] Since no liquid phase reaches the ion source but rather a disperse phase in CO2, APCI and APPI sources often display the best performance in terms of sensitivity and robustness.

Targeted and untargeted lipidomics

Lipidomic studies can be designed according to two approaches, i.e. targeted or untargeted studies. The first aims to extract, enrich and quantify specific lipids from a complex matrix. The second consists of the least selective extraction and large-scale profiling methods of all molecular species present in the sample. Both approaches are well suited to lipidomics because of the wide dynamic range of concentration and the high structural diversity of lipids in tissues, biological fluids or other samples of interest.

Targeted lipidomics methods in SFC-MS have been focused on several classes such as free fatty acids (C18 column, methanol/acetonitrile (50:50, v/v) with 0.1% formic acid) [START_REF] Qu | Direct detection of free fatty acids in edible oils using supercritical fluid chromatography coupled with mass spectrometry[END_REF] or vitamin E derivatives in vegetable oils (NH2 column, pure ethanol), [START_REF] Méjean | Quantification of tocopherols and tocotrienols in soybean oil by supercritical-fluid chromatography coupled to high-resolution mass spectrometry[END_REF] carotenoids in human plasma (C30 column, pure methanol), eicosanoids in human plasma and cerebrospinal fluid (2picolamine column, MeOH with 0.1% formic acid and 10 mM of ammonium acetate), [START_REF] Kumari | A novel, fast and sensitive supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) method for analysis of arachidonic acid metabolites[END_REF] steroids (2-ethylpyridine column, pure isopropanol), [START_REF] Teubel | Methods in endogenous steroid profiling -A comparison of gas chromatography mass spectrometry (GC-MS) with supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS)[END_REF] environmental toxins as acetogenins (2ethylpyridine column, pure ethanol). [START_REF] Laboureur | Structural Characterisation of Acetogenins from Annona muricata by Supercritical Fluid Chromatography Coupled to High-Resolution Tandem Mass Spectrometry[END_REF] These examples perfectly demonstrate the diversity of stationary phase and co-solvent that are described in the literature and which contrast strongly with HPLC studies that lead mainly to the use of C18 columns and water/acetonitrile gradients.

For profiling of complex lipid mixtures, the studies mainly focused on animal and human plasma lipid extracts. It is indeed a good model and a rapid access route to biomedical applications for treatment monitoring or pathology biomarker research. T. Bamba's group has been a pioneer in this field by demonstrating the class separation of a total lipid extract of Madagascar Periwinkle by SFC-ESI-MS. [START_REF] Bamba | High throughput and exhaustive analysis of diverse lipids by using supercritical fluid chromatography-mass spectrometry for metabolomics[END_REF] The authors use a mixture of 14 lipid standards to develop their method. The final chromatographic method requires a cyano column (250 mm x 4.6 mm x 5 µm) and employs methanol with 0.1% ammonium formate as co-solvent. The gradient was programmed over 20 minutes without taking into account rinsing and equilibration times. Several co-elutions were observed, including between phosphatidic acids (PA), phosphotidylethanolamines (PE) and phosphotidylinositols (PI) as well as between phosphatidylserines (PS), phosphatidylcholines (PC) and sphingomyelins (SM). Limits of detection (LODs) were in the range of 60 pmol (PC) to 15,000 pmol (PI) injected. An Inertsil ODS-EP column (250mm x 4.6mm x 5 mm, GL Sciences) was then chosen in 2011 for the separation of ten polar lipid classes after Trimethylsilyl (TMS) derivatization in 10 minutes. [START_REF] Lee | Development of a polar lipid profiling method by supercritical fluid chromatography/mass spectrometry[END_REF] This method led to symmetrical and thin chromatographic peaks, but the derivatization step remains time consuming. Nevertheless, the sensitivity was greatly improved with LODs in the range of 5 (sphingosine-1-phosphate, S1P) to 300 fmol (PA) injected and the method was successfully tested on sheep plasma. The initial underivatized method was then improved in 2012 by using a PC-HILIC column for the separation of polar lipids (PC, PE, SM) from dried plasma spot analysis [START_REF] Uchikata | High-throughput phospholipid profiling system based on supercritical fluid extraction-supercritical fluid chromatography/mass spectrometry for dried plasma spot analysis[END_REF] and in 2018 by a diethylamine (DEA) column and a co-solvent methanol/water (95/5, v/v) with 0.1% formic acid for wide profiling of rabbit plasma lipids. [START_REF] Takeda | Widely-targeted quantitative lipidomics method by supercritical fluid chromatography triple quadrupole mass spectrometry[END_REF] Nineteen lipid classes were detected in less than 20 minutes and 413 species were annotated and semi-quantified according to MS/MS data with limits of quantification (LOQs) from 5 (PC) to 1000 fmol (lysoPA).

M. Holcapek's group also demonstrated the separation of 30 lipid classes from 6 families of lipids (fatty acyles, phospholipids, glycerolipids, sphingolipids, sterols and derivatives, prenols) in less than 6 minutes with a stationary phase BEH silica column (100 mm x 3 mm x 1.7 µm, Waters) and a co-solvent methanol-water mixture (99:1, v/v) containing 30 mM of ammonium acetate. [START_REF] Lisa | High-Throughput and Comprehensive Lipidomic Analysis Using Ultrahigh-Performance Supercritical Fluid Chromatography-Mass Spectrometry[END_REF] By injecting a lipid extract from pig brain, the authors controlled the separation by class by observing ion chromatograms extracted from several species belonging to the same lipid class.

Tests with a similar stationary phase were conducted in our team for the analysis of microalgae extracts but led to the co-elution of glycolipids of interest (monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), diacylglyceryltrimethylhomoserine (DGTS) and sulfoquinovosyl diacylglycerol (SQDG)) with sphingolipids and phospholipids. This observation led us to start a classic method development as briefly described below.

Method development

The set-up available in our laboratory is based on a 1260 Infinity Analytical SFC system hyphenated to a 6540 Q-ToF mass spectrometer (Agilent Technologies, Waldbronn, Germany). A make-up solvent is added between the analytical column and the BPR. The chromatographic flow is then directly introduced to the mass spectrometer through a capillary (500 x 0.17 mm). As previously described by the group of Bamba, [START_REF] Takeda | Widely-targeted quantitative lipidomics method by supercritical fluid chromatography triple quadrupole mass spectrometry[END_REF] we chose a Torus-DEA column (150 mm x 2.1 mm x 1.7 µm) for the method development. The lipid extract of Nannochloropsis gaditana microalgae was provided by J. Jouhet (CEA Grenoble). It was prepared according to the following protocol: the cells were collected by centrifugation and immediately frozen in liquid nitrogen. The pellets were suspended in 4mL of ethanol and boiled for 5 minutes to prevent the degradation by phospholipases. Lipid extraction was then performed by adding 10mL of chloroform/methanol mixture (4:1, v/v) at room temperature.

The solution obtained was shaken for 1 hour at room temperature under argon to avoid any oxidation. After filtration on quartz wool filter, the cells were rinsed with 3mL of chloroform/methanol mixture (2:1, v/v) and 5 mL of 1% NaCl was added to obtain two phases.

The lower phase was sampled and dried under argon. A 1 mg.mL -1 stock solution was prepared in a chloroform/methanol mixture (2/1, v/v) by weighting the extract and was diluted twice before injection.

A generic gradient ranging from 1 to 35% of ethanol was tested in the first approach. This method allows a partial separation of cholesteryl esters with triglycerides, 1,2-and 1,3diglycerides and polar lipids except SQDG. In order to optimize the separation, a 3 min plateau with 15% co-solvent was implemented at 2.5 min elution time allowing a separation with baseline resolution of the different lipid classes. Finally, the percentage of co-solvent at the end of the gradient was increased to 45% allowing the elution of SQDGs and PI which are the most polar species in the mixture. This led to a total time of the SFC run of 20 min including equilibration time. In order to optimize the performance of the SFC-ESI-MS coupling, a set-up with or without split was evaluated by comparing the full widths at half height of the major peaks of the main classes and their peak intensities (Figure S1). The split set-up allows significant refinement of peaks without loss of sensitivity or even a gain, except for the DGs.

The final method leads to peaks with a full width at half height between 4 and 6 seconds (Figure 5).

Once the separation dimension has been optimized, it is necessary to ensure the detection of as many species as possible, whatever their polarity and ionizability. In order to ensure the transfer of analytes to the ESI source of the mass spectrometer and to facilitate their ionization, a make-up solvent is therefore added. After optimizing the solvent composition, we chose to use MeOH/EtOH 1:1 (v/v) as the solvent make-up mixture. We added various additives, such as ammonium acetate at 20 mM, water at 2% (v/v) or formic acid at 0.1% and 0.5% (v/v). Six "make-up" solvents were tested at 3 different flow rates (100, 200, and 400 μL.min -1 ) monitoring the response of one major species per lipid class (Figure S2). For all additives, the compounds that are eluted at the beginning of the gradient are better ionized with a flow rate of 200 μL.min -1 , while the compounds that are most retained are better detected with a lower flow rate of 100 μL.min -1 . In addition, the sensitivity is increased for all lipid classes in positive and negative ionization mode when 20 mM of ammonium acetate are added in the make-up solvent. In that case, ammonium adducts in positive ion mode and acetate adducts in the negative ion mode were mainly detected. Such ionization process led to a better sensitivity for PC in the negative ion mode even if the molecule bears a permanent positive charge on a quaternary ammonium group. Thus the make-up pump will deliver a mixture of MeOH/EtOH 1:1 (v/v) + 20 mM ammonium acetate at 200 μL.min -1 from 0 to 6 minutes, then 100 μL.min -1 from 7 to 19 minutes. It should be noted that the change of the make-up flow during the analysis was not described in the literature and should be compared to the LC-CAD set-up that allows the same solvent composition arriving at the detector to be maintained throughout the analysis.

Finally, the gas temperatures, the voltages applied to the ESI probe and at the fragmentor were optimized in positive and negative ionization mode leading to sensitivities in the order of 10 pg/mg of extracts. A mass spectrum extracted between 0.87 min and 1.79 min is used to annotate up to 34 triglycerides (Figure 6). It should be noted that within the same lipid class, a partial separation of molecular species is demonstrated according to the number of carbons of the fatty chains (Figure S3).

Future Developments

Although the coupling between supercritical fluid chromatography and mass spectrometry has been demonstrated for more than ten years, this technology still faces many challenges in becoming a universal technique like LC-MS. The choice of a large number of stationary phases can be considered as a particularly attractive aspect for carrying out analytical studies but often discourages new users and can be judged as a waste of time and efficiency in an analytical platform context requiring simple and robust methods. Thus, the stationary 2ethylpyridine phase is often viewed as a versatile phase in the literature but is not widely used in the context of lipidomics as previously described. The arrival of new stationary phases based on highly basic material nevertheless opens the way to the gradual removal of additives, i.e. ammonium salts in particular, which require fairly long equilibration and rinsing times for the columns. In the context of lipidomics, it should be noted that the use of chiral stationary phases is rarely described in the literature and deserves particular attention, especially for the separation of arachidonic acid derivatives (leukotriens, prostaglandins, thromboxanes, etc.).

The SFC-MS interface itself still requires further improvements to eliminate peak expansion or molecule condensation phenomena leading to significant sensitivity losses. Nowadays, SFC-MS is still considered less sensitive than LC-MS by one to two orders of magnitude.

Nowadays, APCI and APPI sources, although less popular than ESI sources, appear to be the most suitable for analyzing effluent arriving at the source in the form of a pneumatic spray by expanding CO2 at atmospheric pressure. It is recommended to set up designed experiments to determine the optimal ionization parameters during method development, especially for targeted approaches where trace quantification may be required. As an interlaboratory study was undertaken in SFC-UV, [START_REF] Dispas | First inter-laboratory study of a Supercritical Fluid Chromatography method for the determination of pharmaceutical impurities[END_REF] it would be extremely attractive to carry out the same work with different SFC-MS platforms.

Concerning lipidomics studies in SFC-MS, the pioneering works of T. Bamba and M.

Holcapeck have paved the way for applications in biology, cosmetics or biofuels. The cost of instrumentation and the time required to optimize methods remain a barrier to the dissemination of these methods, which have the advantage of being efficient in terms of separation, fast with analysis times of 20 minutes compared to more than 45 minutes in NPLC for lipid separation by class, and sensitive. Conditions for high-throughput screening are now met and SFC-MS studies should be expanded in this direction. In addition, the adaptation of modern methods for MS/MS annotation of lipids by post-column cationization, [START_REF] Laboureur | Structural Characterisation of Acetogenins from Annona muricata by Supercritical Fluid Chromatography Coupled to High-Resolution Tandem Mass Spectrometry[END_REF] method for labelling double bonds via the Paternò-Büchi reaction [START_REF] Ma | Pinpointing double bonds in lipids by Paternò-Büchi reactions and mass spectrometry[END_REF] or the use of molecular networks for data classification [START_REF] Olivon | MetGem Software for the Generation of Molecular Networks Based on the t-SNE Algorithm[END_REF] have yet to be developed in the near future.

Table 1: Orders of magnitude of physical properties of solvents in different states

Type of fluid

Property

Gas SCF Liquid

Density, r (g.cm -3 ) 10 -3 0.3 1 Diffusivity, D (cm².s -1 ) 10 -1 10 -3 5 x 10 -6

Viscosity, h (g.s.cm -1 ) 10 -4 10 -4 10 -2 Chromatographic column is connected directly to MS through a pressure restrictor heated at its end, (b) same as in (a) but here the restrictor is heated at the beginning, (c) Full flow from the column is first passed through a UV detector, and then is mixed with an organic solvent delivered by a fixed-pressure pump, before being expanded to MS through a restrictor, (d) here flow from the column is passed through an automated back-pressure regulator (ABPR) before the MS and the flow is mixed with a make-up pump flow, either before or after the ABPR, (e) here a part of the outlet from the column is directed towards an MS whereas the other part is taken to an ABPR passing through a UV detector, (f) in this configuration, flow from the column is first passed through a UV detector, and then is mixed with an organic solvent delivered by a make-up pump. A part of the mixed stream is then taken to the MS and the other part to an ABPR.

Figure 1 :

 1 Figure 1: Effect of the addition of ammonium acetate (15 mmol.L -1 ) on the peak shape of polar compounds. Mix of 3 isomers of methylcytidines. Column Waters Acquity UPC² Torus 2-PIC (150 mm x 2.1 mm x 1.7 µm), 150 bar, 30°C; Gradient 5 to 20% of co-solvent (MeOH) in 25 min; Flow rate 1.5mL.min -1 .

Figure 2 :

 2 Figure 2: Spider diagram proposed by West et al. 23 representing a column classification based on quantitative structure-retention relationships (QSRRs). Testing conditions: CO2-MeOH 90:10 (v/v), 25 °C, 150 bar, 1 or 3 mL.min -1 depending on column dimension. Retention data was measured for 85 neutral species with 31 columns.

Figure 3 : 7 Figure 4 :

 374 Figure 3: Schematic view of Infinity 1260 SFC system (Agilent Technologies) and its hyphenation with mass spectrometry. 7

Figure 5 :

 5 Figure 5: UHPSFC/ESI-MS extracted ion chromatogramms in positive (a) and negative (b) ion mode of the major peaks of the main classes of lipid detected in the extract of Nannochloropsis microalgae. UHPSFC conditions: Torus DEA column (150 × 2.1 mm, 1.7 μm, Waters), the flow rate 1 mL.min -1 , the column temperature 60 °C, the ABPR pressure 130 bar and the gradient of methanol-ethanol mixture (1:1, v/v) containing 20 mM of ammonium acetate as the modifier: 0 min, 1%; 1.5 min, 4%; 2.5 to 5.5 min, 15%; 7.5 min, 30%; 8.5 to 15 min; 45%.

Figure 6 :

 6 Figure 6: a) Extracted Ion Chromatogram (EIC) of eight different triglycerides detected in the extract of Nannochloropsis microalgae. b) Extracted Mass spectrum between 0.87 min and 1.79 min.

Acknowledgements

This work was supported by the Agence Nationale de la Recherche (Grant ANR-16-CE29-0002-01 CAP-SFC-MS).


Abdelhakim Bourennane

Adem Lale

Frédéric Richardeau

Monolithic asymmetric switching cells integrated on vertical multi-terminal Si power chips

Monolithic integration », «switching cell», « N-IGBT », « P-IGBT », « PiN diode », « multiterminal Si power chip », « multi-phase Inverter ».

Introduction

Currently commercialized conventional power modules are generally realized using 2D hybrid packaging technology in which two-terminal power dies such as Insulated Gate Bipolar Transistor (IGBT) and PiN-diode are bonded on their backside and interconnected using wire bonds. A power module is made of a great number of two-terminal dies that are interconnected by wire bonds. Power modules are therefore bulky and their mass fabrication is limited in productivity. Moreover, wire bonds in power modules are source of parasitic inductance [START_REF] Tanimoto | High junction temperature and low parasitic inductance power module technology for compact power conversion systems[END_REF][START_REF] Li; Tolbert | Stray Inductance Reduction of Commutation Loop in the P-cell and N-cell-Based IGBT Phase Leg Module[END_REF] that induces high turn-off voltage overshoot, which not only add electrical stress on the power dies, but also result in switching losses and thus affect the overall efficiency. Another undesirable phenomenon regarding the 2D standard power modules concerns the common mode currents. Indeed, the mid-point of each phase-leg is at a floating potential that evolves as function of the high dv/dt during switching phases, the worst cases being well-known: at low load-current turn-on and at high load-current turn-off. The mid-point being electrically contacted to the substrate through wire-bonds generates common-mode currents through the parasitic capacitors resulting from the succession of the layers that form the circuit board and the heat-sink. It is important to limit the amplitude of this current through the use of common mode filters.

To improve reliability, electrical performance and reduce cost of power modules, significant breakthrough can be achieved by the monolithic integration of the 2D-multiphase module within a minimum number of multi-terminal dies that are judiciously packaged. Different approaches for converter integration were described in literature in order to reach similar objectives. However, they are either limited in performance (lateral integration with vertical insulation trenches [START_REF] Green | Fully Isolated High Side and Low Side LIGBTs in Junction Isolation Technology, Power Semiconductor Devices and IC's, ISPSD[END_REF]) or require a complex technological process for their realization (molecular bonding of two wafers [START_REF] Benaissa | A vertical power device conductive assembly at wafer level using direct bonding technology[END_REF]). Within the same context, the authors have recently proposed vertical quasi-monolithic integration approaches of power converter [START_REF] Khadiry | Multiphase Power Converter Integration in Si: Dual-Chip and Ultimate Monolithic Integrations[END_REF][START_REF] Abdelhakim | Multi-pole power chip(s) with monolithic integration of asymmetric chopper cells and multi-phase power module(s) using the or a plurality of said multi-pole chips, Priority number FR20160063038 20161221[END_REF][START_REF] Lale | Monolithic Complementary Multi-terminal RC-IGBT Chips for Compact Multi-phase Power Converter[END_REF][START_REF] Lale | Realization of a monolithic multiterminal Si-power chip integrating a 2-phase rectifier composed of vertical PiN diodes insulated by vertical P+ walls[END_REF][START_REF] Lale | Mixed monolithic-hybrid integration of multiphase power converter : Preliminary evaluation of the 3-chip integration concept[END_REF].

Generally, the adopted strategy for integrating multi-phase power converters resides on a fractionated integration of the switching cell with a full separation between the high-side and the lowside switches of the converter [START_REF] Khadiry | Multiphase Power Converter Integration in Si: Dual-Chip and Ultimate Monolithic Integrations[END_REF][START_REF] Abdelhakim | Multi-pole power chip(s) with monolithic integration of asymmetric chopper cells and multi-phase power module(s) using the or a plurality of said multi-pole chips, Priority number FR20160063038 20161221[END_REF][START_REF] Lale | Monolithic Complementary Multi-terminal RC-IGBT Chips for Compact Multi-phase Power Converter[END_REF][START_REF] Lale | Realization of a monolithic multiterminal Si-power chip integrating a 2-phase rectifier composed of vertical PiN diodes insulated by vertical P+ walls[END_REF][START_REF] Lale | Mixed monolithic-hybrid integration of multiphase power converter : Preliminary evaluation of the 3-chip integration concept[END_REF]. Indeed, the high-side part of the converter is integrated in one multi-terminal power chip while the low-side part is integrated in another multi-terminal power chip. Even though the followed strategy permits to minimize the number of multi-terminal chips for the converter realization, the functionality of the elementary switching cell can only be synthesized by interconnecting at least two power chips. The new approach proposed in this paper aims at integrating monolithically the switching cell, in a buck or boost mode, within a single silicon chip. The realization of a phase-leg, unidirectional in current, requires consequently only one chip. The current bidirectionality, which is required in the case of an inverter, necessitates the use of two chips per phase-leg. The new proposed approach allows realizing a monolithic switching cell with an intrasilicon switching loop that one can qualify as the ultimate loop.

The paper focuses on the ultimate monolithic switching loop. Its operating modes are validated by Sentaurus TM 2D simulations and the optimized packaging of the chips is discussed.

The concept of ultimate wire-bond-less monolithic switching cell in a silicon substrate

The concept of integrating asymmetric cells is illustrated in Fig. 1 and described in [START_REF] Abdelhakim | Multi-pole power chip(s) with monolithic integration of asymmetric chopper cells and multi-phase power module(s) using the or a plurality of said multi-pole chips, Priority number FR20160063038 20161221[END_REF]. It aims at integrating the voltage inverter-leg within two macro-monolithic chips. One chip that monolithically integrates the high-side IGBT with the low-side diode named a buck-switching cell and the other integrates the low-side IGBT with the high-side diode and is named a boost-switching cell. The boostcell is integrated in an N-substrate while the buck-cell is integrated in a P-substrate. The gates of the IGBTs are controlled with respect to two constant reference potentials imposed by the external power supply. The gate of the P-IGBT is controlled with respect to VDC while the gate of the N-IGBT is controlled with respect to ground. As a result, the power supplies of the gate drivers are not polluted by mid-points dv/dt at switching, ensuring thus a safer operation. These reference electrodes allow sharing the power supply of the different gate drivers that are referenced to the same electrode. Consequently, only two power supplies are required for the gate drivers of a multiphase converter. This reduces the complexity of the power circuit, reduces the costs and improves reliability. region of the IGBT. This backside P + region is also used as a mechanical support for the partial vertical N + wall for lateral electrical insulation. Fig. 3 shows the monolithic structure of the buck switching cell integrating a P-IGBT and a P-diode on a P-epitaxial substrate. The backside N + region is used as the cathode region of the diode and the emitter region of the IGBT. This backside N + region is also used as a mechanical support for the partial vertical P + wall for lateral electrical insulation. Each chip is a three-terminal chip (3 power electrodes): the DC + and DC -electrodes that are placed on the front-side are the two electrodes of the external power supply while the third electrode placed on the backside is the phase-leg mid-point. One can notice that no wirebonds were required to realize the switching cell. Consequently, this switching cell can be qualified as being the ultimate switching cell.

To realize a complete voltage inverter phase-leg, the two complementary chips need to be assembled. Fig. 4 shows a possible packaging of the two complementary monolithic switching cells. The chips are bonded by their backside in a conventional manner. By analyzing this assembly, one can remark that the fact that the electrodes DC+ and DC-are on the front-side make easier their connection to the busbar using a unique connection like a (ribbons, integrated busbar, copper clip or direct lead bonding). Such interconnect permits to minimize stray inductance between high voltage DC supply and the power chips. A second interesting property concerns the possibility of placing a decoupling capacitor above each chip. Gate drivers can also be placed above the chips on the same horizontal plane of the busbar. This can be achieved by gathering the drivers and the busbar on the same PCB that will be placed above the chips.

One drawback of this packaging resides in the placement of the phase-leg mid-point (PM). Indeed, this latter is bonded on the PCB substrate. The voltage of this mid-point voltage varies according to the commutations dv/dt and is a source of the undesirable common mode parasitic current that flows through the capacitive layers of the PCB substrate. An alternative packaging to overcome this drawback would consist in flipping the chips but that leads to an increase in the complexity of packaging technology process (not studied in this paper).

Validation of the operating modes of the ultimate wire-bond-less single switching cell

The operating modes of the two monolithic ultimate switching cell were analyzed using Sentaurus TM 2D simulations. The active area of each switch section within each multi-terminal chip is 1cm 2 for a 600V rating. Each chip is first simulated separately within a chopper circuit application and then they were assembled to form a full voltage inverter phase-leg.

Basic boost switching cell

The monolithic boost switching cell was simulated during a full switching cycle. The two conducting modes of the switching cell were analyzed by observing current density distribution as well as equipotential lines within the structure. The main objective is to check that when one section is in conducting state, the other is in off-state and supports the applied DC bus voltage with a negligible leakage current.

Basic buck switching cell

The buck cell is also simulated in a chopper circuit. Fig. 8 shows the simulation results for the IGBT mode and Fig. 9 simulation results for diode mode. In IGBT mode, one can notice that the current flowlines flow from the anode electrode (frontside) towards the midpoint in the backside. The space charge region in the left-side section shows that the diode section is reverse biased and supports the applied voltage. This space charge region corresponds to the reverse biasing of the junction P-drift

Fig. 1 .

 1 Fig. 1. Illustration of the monolithic switching cell concept.

Fig. 2

 2 Fig.2shows the monolithic structure of the boost switching cell, integrating an N-IGBT and a diode on N -substrate. The backside thick P + region is used as the anode region of the diode and as a collector

  IGBT high-side + diode low-side P-wafer Monolithic boost cell IGBT low-side + diode high-side N-wafer Load

Fig. 2 .Fig. 3 .

 23 Fig.2. (a) 2D cross sectional view of the monolithic wireless boost cell and (b) electrical circuit.

Fig. 4 .

 4 Fig. 4. Proposed assembly of the inverter phase-leg consisting of two complementary cells: (a) version with decoupling capacitors above the chips and (b) version with decoupling capacitors placed on the sides of the chips.

Fig. 5

 5 Fig.5shows 2D simulation results for IGBT mode. Fig.6shows 2D simulation results for diode mode. In the IGBT mode, one can notice a high current density in the right-most IGBT section and decreases as one moves towards the diode section. The current flowlines flow from the backside midpoint region towards the emitter electrode of the IGBT in the front-side. The DC-bus voltage is supported by the diode section and a space charge region expands within the N-drift region of the diode. The junction P + wall/N-is reverse biased. In the diode mode, one can notice a high current density in the left-most section of the chip. The mid-point voltage is almost at the DC-bus voltage and therefore the space charge region expands within the N-drift region.

Fig. 6 .

 6 Fig. 6. (a) Diode mode of the boost cell, (b) current density and equipotential lines in diode mode.

Fig. 7 .

 7 Fig.7. (a) Current and voltage waveforms within a commutation cycle of the boost cell and (b) zoom into the dynamic phases of commutation.

Fig. 9 .

 9 Fig. 9. (a) Diode mode of the buck cell, (b) current density and equipotential lines in diode mode.

Fig. 10

 10 Fig.10 shows voltage and current waveforms in the buck cell during one switching cycle. A zoom into the dynamic phases given in Fig. 10(b) allows a better analysis of the commutation sequences. At IGBT turn-on, one can notice the diode reverse recovery current (red curve) and the mid-point voltage switches from DC-voltage (about 0V) to DC+ voltage (about 300V). At IGBT turn-off, the mid-point switches back to DC-voltage (about 0V). These waveforms are similar to those of the classical buck cell and allow to validate the operating modes of the monolithic buck switching cell. The comparison of the diodes post-recovery current waveforms of Fig. 8(b) and Fig.10(b) show that the P-diode postrecovery current is more abrupt than that of the N-diode. Additional 2D simulations will be carried-out in order to confirm this difference. Practically, the snappy recovery is a source of overvoltage across the diode and EMI. Solutions based on a snubber circuit or a clamp placed in parallel with the diode can be used. The control of the converter in ZVS mode also permits to overcome these problems.

This research work received financial support from the French National Research Agency (ANR). The project name is "ConvPlUS", ANR-13-JS09-0008-01, using the technological support of RENATECH-LAAS and 3DPHI platforms.

Association of the elementary cells in an inverter phase leg

The functionality of the phase leg inverter is obtained by associating the two elementary complementary cells as shown in Fig. 11.

Conclusion

In this paper, a concept of asymmetric monolithic switching cell integration is presented and validated by 2D physical simulations. The concept relies on the integration of the inverter phase-leg on two complementary chips, in which each chip integrates a monolithic switching cell. The first chip integrates the buck cell within a p-wafer while the second chip integrates the boost cell in an N-wafer. The operating modes of each cell were first validated separately and then associated in a phase-leg's inverter application. This is the ultimate monolithic wire-bondless switching cell that allows to suppress the wirebonds within the switching cell and therefore minimizes the stray inductance value. Consequently, it is suited for the emerging applications requiring to achieve simultaneously: reliability, compactness, intrinsic performance and reduced manufacturing cost.