Design of Integrated Laser Diode Driver for 3D-Depth Sensing Applications

This paper presents the design and comparison of two integrated CMOS Laser Diode Drivers (LDD) intended for 3D-depth sensing applications used in mobile phones and based on the indirect Timeof-Flight (iToF) measurement. Architectures are based on known driver topologies where a standard DC/DC converter is connected to a switching element either in series or parallel to provide pulses. Two LDD prototypes have been implemented on-chip using a 130nm CMOS technology from STMicroelectronics. Both prototypes are able to generate current pulses up to 3A with a 2.5ns pulse width at a maximum 200MHz Pulse Repetition Frequency (PRF) under a 3.6V supply voltage. In these conditions, measurement results report an average output electrical power of 4.5W with an electrical efficiency of 58% and 62% for series and shunt configuration respectively.

Introduction

Nowadays, emerging applications such as facial recognition, gesture control or Augmented Reality (AR) are in great demands in consumer electronics [START_REF] Lineback | A Market Analysis and Forecast for Optoeletronics, Sensors/Actuators, and Discretes[END_REF]. Developing a 3D image sensor able to map the surrounding area in three dimensions would benefit these applications. The indirect Timeof-Flight (iToF) measurement technique offers the advantage of a straightforward, robust and low cost implementation for mobile applications by using a laser transmitter and an image sensor [START_REF] Stoppa | Scannerless 3D imaging sensors[END_REF] [START_REF] Suss | Benchmarking time-of-flight based depth measurement techniques[END_REF]. Its operating principle is to calculate a depth by measuring the phase shift between a modulated infrared laser signal and the optical signal received by the sensor after reflection on an object from the scene. The block diagram of a typical iToF-based image sensor is illustrated in Figure .  Generally, laser pulses with a duty cycle close to 50% are sent through the scene as the modulated signal, corresponding to the Continuous Wave (CW) modulation [START_REF] Niclass | Single-Photon Image Sensors in CMOS: Picosecond Resolution for Three-Dimensional Imaging[END_REF]. A high optical power of laser pulses as well as a high Pulse Repetition Frequency (PRF) are desired to improve the depth resolution in iToF systems, as defined by

∝ 4 √ 2 ⋅ √ (1)
where is the speed of light, is the PRF and is the peak optical power at the laser emitter level [START_REF] Lange | Solid-State Time-of-Flight Range Camera[END_REF]. Moreover, multiple frequencies are needed to extend the maximum unambiguous range, due to the phase shift wrapping every 2 , without impacting the measurement accuracy [START_REF] Jongenelen | Maximizing precision over extended unambiguous range for TOF range imaging systems[END_REF].

Depending on the maximum targeting depth, lighting conditions, object reflectivity, sensor efficiency, the choice of the laser diode and eye-safety standards, current pulses up to few Amps at a frequency from tens to hundreds of MHz are required to reach a millimeter depth accuracy at few meters of distance. However, short pulse trains are needed to avoid thermal dissipation issues and human eye damages according to laser safety standard [START_REF]Safety of laser products -Part 1: Equipment classification and requirements[END_REF]. These specifications introduce new challenges in the design of a compact, low-cost and efficient Laser Diode Driver (LDD). Main concerns are the high level of current and the shaping of short-duration pulse at high repetition frequency because of the parasitic elements in passive components and PCB interconnections. In addition, mobile applications bring strong constraints in design choices such as dealing with the restricted available space and accommodating the limited input voltage range of a battery (2.5-4.8V). Finally, a high efficiency in the chip is critical for saving the battery lifetime and minimizing any self-heating. This paper focuses on the design and comparison of two known driver topologies: series and shunt what means the laser diode is connected either in series or parallel with a switching element in order to provide current pulses. In addition, a standard DC/DC converter is used for controlling the current through the laser diode with a high efficiency.

The novelty here concerns the feasibility of integrating the whole solution (except laser diode and passive components). Current pulses can be configurable up to 3A peak for a PRF from 50MHz to 200MHz with a 50% duty cycle under typical conditions. Section 2 provides a quick review on various circuits from the literature. Section 3 explains the operating principle of the proposed architectures for the LDD. Section 3 describes the on-chip implementation of the two integrated LDD prototypes. Experimental results performed on both prototypes with a laser diode are reported in Section 4. Finally, last section discusses future works and concludes the paper.

LDD Topology Background

Several works have been published presenting LDDs for ToF applications with various current, pulse width and PRF specifications. Drivers may be divided into two major classes according to the classification described in [START_REF] Glaser | How GaN Power Transistors Drive High-Performance Lidar: Generating ultrafast pulsed power with GaN FETs[END_REF]. The classification depends on how the pulse is controlled. There are resonant capacitive discharge drivers and rectangular pulse drivers.

Resonant capacitive discharge drivers

Although several configurations can be distinguished from circuits in [START_REF] Nissinen | A 1A Laser Driver in 0.35µm Complementary Metal Oxide Semiconductor Technology for a Pulsed Time-of-Flight Laser Rangefinder[END_REF][10] [START_REF] Hallman | A high-speed/power laser transmitter for single photon imaging applications[END_REF][12] [START_REF] Glaser | High Power Nanosecond Pulse Laser Driver using a GaN FET[END_REF], they are based on the same operating principle firstly proposed by Kilpelä et al. in [START_REF] Kilpelä | Laser pulser for a time-of-flight laser radar[END_REF]. A schematic is shown in Figure 2 in a basic version. This type of drivers rely on a capacitive discharge through a switch and laser diode to generate a pulse by triggering the rising edge only.

This topology has a relatively simple design accepting the stray inductance into its operation. The laser pulse energy is well controlled, thus eliminating thermal runaway and providing an improved pulse-to-pulse repeatability [START_REF] Glaser | How GaN Power Transistors Drive High-Performance Lidar: Generating ultrafast pulsed power with GaN FETs[END_REF]. However, a major drawback concerns the pulse width that depends on the capacitor and stray inductance, which are fixed for a given design, while the pulse amplitude depends on the supply voltage and the capacitor too [START_REF] Nissinen | A High Repetition Rate CMOS Driver for High-Energy Sub-ns Laser Pulse Generation in SPAD-Based Time-of-Flight Range Finding[END_REF]. Therefore, it is impossible to control the pulse width and amplitude separately. Another issue is the long time needed to charge the capacitor thus limiting the PRF. Moreover, a relatively high supply voltage may be required to achieve a high peak current what may increase the design complexity, cost and space needed by the whole circuit.

Rectangular pulse drivers

Rectangular pulse drivers generates current pulses by triggering both the rising and falling edges. Then, two types of driving mode can be distinguished for controlling the laser diode biasing: the voltage-mode driver based on a voltage source or the current-mode driver based on a current source.

Driver circuits presented in [13][15] are considered here as voltage-mode drivers using a series configuration, as illustrated in a basic version in Figure 3a. A voltage source is connected in series to a laser diode and a switch. This topology benefits from a control of the pulse width and a high PRF can be reached due to the use of a relatively large decoupling capacitor [START_REF] Glaser | How GaN Power Transistors Drive High-Performance Lidar: Generating ultrafast pulsed power with GaN FETs[END_REF]. Moreover, the peak current is independent of the pulse-width setting and only a control of the laser diode biasing voltage is required to adjust its value. These features allows flexible pulse sequences. However, the supply voltage needs to be high enough to overcome the stray inductance. Furthermore, control loops may be required to ensure pulse-to-pulse repeatability due to temperature and voltage variations [START_REF] Ma | A Digital-Type GaN Driver with Current-Pulse-Balancer Technique Achieving Sub-Nanosecond Current Pulse Width for High-Resolution and Dynamic Effective Range LiDAR System[END_REF].

Other circuits reported in [16][17] are considered here as current-mode drivers using a current source configuration, as illustrated in a basic version in Figure 3b. A laser diode is connected in series to a low-side current source and a switch, thus acting as a switched current generator. A voltage source is still used to provide a constant voltage to the laser diode. Even if flexible pulse sequences can be achieved with a high PRF, a major limitation concerns the relatively high dissipated power through the switched current generator when turned on due to the sufficient voltage headroom required for its proper biasing [START_REF] Blasco | A Sub-ns Integrated CMOS Laser Driver With Configurable Laser Pulses for Time-of-Flight Applications[END_REF].

An alternative approach developed in [START_REF] Abramov | Low Voltage Sub-Nanosecond Pulsed Current Driver IC for High-Speed LIDAR Applications[END_REF] is considered here as a current-mode driver using a shunt configuration, as illustrated in a basic version in Figure 3c. A current source is connected to a laser diode and an assembly of three switches to provide current pulses by shunting the current from the laser diode to the ground. As for previous configurations, flexible pulse sequences with a high PRF can be considered. Nevertheless, whereas several switches are used allowing a more precise control of the current pulse width, it contributes to increase the power consumption and chip size due to additional gate drive circuits.

The circuits considered in this short review are mostly intended for LiDAR applications where constraints in term of power losses are more flexible than for mobile applications and the technologies used might also be specific and incompatible with a low cost requirement, such as GaN FET in [START_REF] Glaser | High Power Nanosecond Pulse Laser Driver using a GaN FET[END_REF] [START_REF] Ma | A Digital-Type GaN Driver with Current-Pulse-Balancer Technique Achieving Sub-Nanosecond Current Pulse Width for High-Resolution and Dynamic Effective Range LiDAR System[END_REF], BCD in [START_REF] Tajfar | A 20 A Sub-Nanosecond Integrated CMOS Laser Diode Driver for High Repetition Rate SPAD-Based Direct Time-of-Flight Measurements[END_REF] or LDMOS in [START_REF] Abramov | Low Voltage Sub-Nanosecond Pulsed Current Driver IC for High-Speed LIDAR Applications[END_REF]. However, the voltage-mode driver topology in series configuration as well as the current-mode driver topology in shunt configuration seem to be good candidates for developing a low-cost integrated driver in standard 130nm CMOS technology able to generate short and high current pulses at a high PRF while operating at a low input voltage range and considering the power efficiency as a leading requirement.

Driver Architectures and Operating Principle

Voltage-mode Driver in Series Configuration

A simplified schematic of the proposed architecture for the voltage-mode driver in series configuration is illustrated in Figure 4. It consists of a buck-boost DC/DC converter and a switch connected in series to the laser diode. This DC/DC converter acts as a current-controlled voltage source for biasing the laser diode by adjusting the output voltage according to the input voltage and the desired current. The switch is triggered by a modulation signal in order to generate current pulses. The current-control loop actually monitors the average current through the laser diode while the peak current value is controlled by the duty cycle of the modulation signal, approximated as

= (2)
The switching frequency of the converter can be selected sufficiently low (<10MHz) compared to the PRF (50MHz-200MHz) in order to benefit from relatively low switching losses and some flexibility for implementing the feedback loop. The output voltage provided by the converter is thus seen relatively constant during the pulse duration leading to a stable current pulse. A power inductor with low value (L OUT ∼ hundreds of nH) and an output capacitor with high value (C OUT ∼ µF) can be selected to ensure a low output voltage ripple.

PWM I REF

High frequency modulation

Current sensing

L OUT I OUT V IN BUCK-BOOST DC/DC POWER STAGE C OUT V OUT

Current-mode Driver in Shunt Configuration

A simplified schematic of the proposed architecture for the current-mode driver in shunt configuration is illustrated in Figure 5. It consists of a modified buck DC/DC converter and a switch connected in parallel to the laser diode. This buck DC/DC converter is used without output capacitor. It thus acts as a current-controlled current source for biasing the laser diode by adjusting the output current according to the input voltage and the desired current. The switch is triggered by a modulation signal in order to shunt the current to the ground thus generating current pulses. In contrast to the previous voltage-mode driver architecture, the currentcontrol loop monitors the average current through the inductor which corresponds to the peak current through the laser diode. The switching frequency of the converter can be selected sufficiently low (<10MHz) compared to the PRF (50MHz-200MHz) in order to benefit from relatively low switching losses and some flexibility for implementing the feedback loop. The output current provided by the converter is thus seen relatively constant during the pulse duration leading to a stable current pulse. A relatively high value for the power inductor (L OUT ∼ µH) is also necessary to maintain a low inductor current ripple.

IC Implementation

This section details the design and implementation of two integrated LDD prototypes based on the architectures previously described. An integrated solution is essential in order PWM I REF

High frequency modulation

Current sensing to develop a compact, fast and low cost system. Indeed, it allows to overcome parasitic elements due to packages and interconnections thus improving transient performances, reducing losses and saving PCB footprint compared to a discrete approach. The selected 130nm CMOS technology employed here is inexpensive and well adapted to embed analog, digital and power features into a single chip. Figures 6a and6b show a block diagram of both voltagemode and current-mode drivers. In order to save time and design resources, a non-inverting buck-boost DC/DC converter has been reused from previous developments to implement the DC/DC converter required by the voltage-mode driver topology. Although made of a specific architecture, it can be seen as a block able to step up or step down the output voltage according to the desired current. A more standard buck DC/DC converter, without output capacitor, has been reused as well to implement the DC/DC converter required by the current-mode driver topology. Both converters employ a standard Pulse Width Modulation (PWM) method using a current-control loop in order to adjust the output voltage according to the desired current value set by a currentlevel reference, as described in [START_REF] Lee | A Monolithic Current-Mode CMOS DC-DC Converter With On-Chip Current-Sensing Technique[END_REF]. The converter architectures and their operating principles are not detailed here. The main contribution of this work concerns the design of the High Frequency Switching (HFS for short) block which aims to switch the current through the laser diode at a high frequency. Furthermore, only one HFS block has been designed since it has similar specifications for both drivers.

L OUT I OUT V IN

HFS Block

A thick oxide n-channel MOSFET transistor (NMOS), named T SW , ensures the current switching at high frequency. It withstands voltages up to 4.8V. Since the peak current (up to 3A) and the PRF (50MHz-200MHz) are flexible, the gate width of the power transistor T SW has been made programmable and divided into 16 equal parts for optimizing both conduction and switching losses. It is configurable here from 5.4mm to 86.4mm by step of 5.4mm. This transistor is driven by an external differential modulation signal through an appropriate driving circuitry. It consists of a simple tapered buffer and a Low Voltage Differential Signal (LVDS) receiver used to convert the fast differential signal (MOD P and MOD N inputs) into a single-ended modulation signal. A cascode transistor, named T CASC , as well as a clamping diode are used to protect devices at the switching node V SW from high voltage spikes due to parasitic inductances occurring during fast transient currents. Transistor T CASC has been implemented using a thick oxide NMOS device with a 24mm gate width sized by considering a trade-off between its onresistance and output capacitance. The additional clamping diode D CLAMP is a typical P-N junction and has been sized large (458x71µm 2 ) enough to evacuate the residual energy from parasitic inductances.

Layout Considerations

Some layout considerations have been employed such as using isolation wells and guard rings in order to isolate the bulk of power devices from the substrate. It helps to reduce substrate coupling and minimize latch-up susceptibility due to carrier injection during fast transients. A dedicated power supply path with an assigned pin for each analog, digital and power domains has been used for minimizing the impact of noise between each block. Higher levels of metal with a large number of Vertical Interconnect Access (VIAs) in parallel are used for power routing. An integrated Metal-Insulator-Metal (MIM) capacitance, named C DEC , has been implemented in the remaining silicon area for an effective decoupling of the buffer power supply, thus reducing the voltage ripple. For easy readability, only the physical layout of the HFS block (without the clamping diode because lo- cated further in the chip) is illustrated in Figure 7. The overall silicon area for the voltage-mode and current-mode LDD prototypes are around 7.25mm 2 and 6mm 2 respectively.

Simulation Results

Post-layout simulations with parasitic models of package and PCB have been carried out with a SPICE simulator to validate the circuit implementation and to evaluate performances of the LDD prototypes. A Vertical-Cavity Surface Emitting Laser (VCSEL) array diode module has been chosen as load with respect to specifications. Main electro-optical characteristics are summarized in Table 1. The Power Conversion Efficiency (PCE) is defined as the output optical power over input electrical power ratio of the VCSEL module. The slope efficiency is defined as the output optical power over input current ratio of the VCSEL module. These parameters are only given at 25°C room temperature due to lack of data from VCSEL suppliers. An electrical SPICE-based diode model is considered for simulation, stating a 2.7V forward voltage for reaching a 3A peak current. The equivalent stray inductance in the laser diode and HFS block current paths has been evaluated at 1.5nH for the voltage-mode driver (as a series configuration is employed). For the current-mode driver, the stray inductance has been evaluated at 800pH through the laser diode current path and 700pH through the HFS block current path (as a shunt configuration is employed). Results for a 3A peak current with a 2.5ns pulse width at 200MHz under typical conditions (typ. process, 3.6V input voltage, 25°C room temperature) are summarized in Table 2. The output power accounts for the average power provided by the driver to the laser diode module during several modulation periods in steady-state while the input power is the average power provided by the power supply to the chip during the same time period. The electrical efficiency is the output power over input power ratio. The dissipated power in IC accounts for total losses due to the integrated circuit only, not including off-chip passive components. The gate width of transistor T SW has been properly chosen for minimizing total losses. Transient waveforms of interest are illustrated in Figure 8 and 9 for the voltage-mode and current-mode drivers respectively.

Although a proper gate signal is provided to the power transistor T SW , a strong distortion on the laser diode current is noticed. It is caused by the strong parasitic inductances, where the rising time is slower than the falling time for the voltage-mode driver while this is the opposite for the current-mode driver. This particularly impacts the currentmode driver where the current is not totally shunted at the end of a pulse bringing an offset that causes the average current through the laser diode to increase, as noticed in Table 2.

The average current regulation for the voltage-mode driver is disturbed as well, leading to a higher peak current (3.5A) than expected, what is explained by the clamping diode inducing a low reverse current.

Voltage spikes are noticed at the switching node in each case, what seems to be clamped to some extent by the clamping diode. Even if over voltages above 4.8V are reported, the cascode transistor T 2 still ensures a protection for the power transistor. It can also be noticed that a 5V output voltage against a 3.6V input voltage is required for the voltage-mode driver to reach the desired current, confirming that a buckboost structure is essential for this kind of driver topology with respect to specifications.

Finally, whereas efficiency values around 60% are reported, the figures seem reasonable in typical conditions at first order. In addition, although parasitic inductances have a strong impact on transient performances, simulation results validate the circuit implementation, proving that both drivers are able to generate high current pulses at high frequency. The equivalent stray inductance could be reduced with a proper PCB design.

Experimental Results

Measurement setup

A test bench has been set up for performing electrical and optical measurements, as shown in Figure 10. The VCSEL module and each LDD prototype were assembled on a test board with optimized interconnections for easy configuration and improved measurements.

Figure 10a shows a Keysight N6705B DC power analyzer to generate the power supply, and a Agilent 81110A pulse generator as well as a Keysight 33600A waveform generator to respectively generate the differential signal (LVDS) and an envelope signal providing pulse trains. Figure 10b shows a Newport 819D-SL-5.3-CAL2 integrating sphere. Associated with a Newport 918D-SL-OD1R optical power detector and a Newport 1936-R optical power meter, this setup has been used for measuring average values of the optical power associated with a pulse train. The peak optical power P PEAK of pulses is determined knowing the duty cycle of the laser signal, such as

= ( 3
)
where P AVG is the average optical power over a pulse train. Figure 10c shows a iCHaus ic212 high-speed free-space photodetector with a 1.4GHz bandwidth to acquire the laser pulse shape. Signals are measured with a Keysight MSOS-254A 2.5GHz mixed-signal oscilloscope. A short illumination pattern has been set to reduce self-heating effects as much as possible. Finally, a STM32F407 microcontroller from STMicroelectronics has been used for configuring the integrated circuit.

Primary results and EMI issues

Primary electrical results have reported some interferences on an analog block within the chip while the current is switching (during a pulse train). This block is a voltage reference providing a fixed voltage of 1.22V used for the current-control loop. The bandgap voltage has been directly measured through a dedicated output pin for both the voltage-mode and current-mode drivers for various desired peak current and frequencies under typical conditions (3.6V input voltage, 25°C room temperature). Results, presented in Figure 11, show that the bandgap voltage decreases as the peak current and frequency increase thus impacting the current regulation. The bandgap voltage drops down to 1.15V and 1.08V for the voltage-mode and current-mode drivers respectively while a 1.22V voltage level is expected. These interferences are probably due to a ground coupling between the HFS block and the bandgap circuit that may arise from interconnections through the substrate of the chip, metallization, PCB tracks and/or ground plane. Despite these EMI issues, the circuit can still be used in an open-loop manner by adjusting the desired peak current value. Following performance results are thus obtained in open-loop.

Performance results and comparison

Both prototypes have been measured using similar conditions in order to make a fair comparison. Since the current regulation may be disturbed due to EMI issues on the bandgap circuit, the peak current value was swept until reaching a desired peak optical power. The pulse width of the external differential modulation signal has been finely tuned to face a potential laser signal distortion. A 50% modulation duty cycle on the laser pulse shape was targeted. Then, transient performances such as rising and falling times, and electrical efficiency have been measured for comparison. The electrical efficiency is defined as

= ( 4
)
where P IN is the driver input power and P OUT is the average electrical output power provided by the driver to the VC-SEL module during a pulse train. Since the output current cannot be measured, P OUT has been estimated from optical power measurements using the VCSEL PCE (see Table 1). The peak current value has been estimated using the VCSEL slope efficiency (see Table 1). Figure 13: Transient measurement waveforms for the currentmode driver for a 2.5W peak optical power with a 2.5ns pulse width at 200MHz under typical conditions. The laser pulse shape is normalized on y-axis according to its maximum value. Important: electrical and optical waveforms are not in phase.

Transient measurement waveforms including the laser pulse shape are shown in Figure 12 and Figure 13 for the voltage-mode and current-mode LDD prototypes respectively. They are obtained while operating under typical conditions (3.6V input voltage, 25°C room temperature) for a targeted 2.5W peak optical power with a 2.5ns pulse width at 200MHz. The gate width of the power transistor has been properly chosen to minimize losses. Performance results are summarized in Table 3.

It can be noticed that both prototypes are able to generate laser pulses. A 4.1V output voltage is reported for the voltage-mode driver, indicating that a buck-boost structure is essential for this kind of topology with respect to the specifications. Slight voltage bounces are however observed on Moreover, even if a pulse width of around 2.5ns is obtained for both prototypes, it can be seen that the pulse shape is slightly distorted. Falling time is slower for the currentmode prototype than for the voltage-mode prototype while it is the opposite for the rising time, confirming simulation results. This is due to parasitic elements and mainly parasitic inductances. In addition, a very short secondary laser pulse occurs when the pulse is cut off for the voltage-mode driver. It might be caused by the strong voltage ringing at the switching node during transient which faded sufficiently to let a reactivation of the laser diode. A slight overshoot is also noticed on the laser signal when the pulse is turning on in the current-mode driver. This might be caused by the strong voltage ringing at the switching node.

For 2.5W peak optical power, it has been evaluated a 62% and 58% electrical efficiency for the current-mode and voltage-mode driver prototypes respectively. It suggests that the current-mode driver in shunt configuration could be more efficient than the voltage-mode driver in series configuration in the proposed measurement conditions. However, no strict conclusion can be safely drawn as some issues have to be solved, such as EMI issues.

Future Works and Conclusion

Two integrated laser diode driver prototypes based on different driver topologies have been developed and implemented on-chip using a 130nm CMOS technology. A test bench has been set up for performing electrical and optical measurements. It has been shown that both drivers are capable of generating laser pulses for a 2.5W peak optical power with a 2.5ns pulse width at 200MHz under typical conditions (3.6V input voltage, 25°C room temperature). In these conditions, a relatively high efficiency is noticed which is quite satisfying in the context of mobile applications. Unfortunately it may not be compared to the state-of-art circuits as efficiency values are rarely mentioned. Indeed, they are mostly intended for LiDAR applications where constraints in term of power losses are more flexible than for mobile applications.

However, experimental results do not fully demonstrate the feasibility of integrating these kinds of driver architecture on a single chip taking into account mobile phone constraints. Further investigations are required, such as investigating EMI issues and performing more measurements while sweeping a wide range of parameters (frequency, peak optical power, input voltage, temperature) to get exhaustive results. This calls for additional PCBs and a second set of IC prototypes. The experimental setup could be consolidated as well to ease the measurement procedure and improve accuracy wherever it is possible. Validating the IC prototypes at system level by considering a typical Time-of-Flight application is also essential in order to demonstrate optical performances of the two different driver topologies.

Figure 1 :

 1 Figure 1: Block diagram of a typical iToF-based image sensor. The depth formula is indicated, where is the phase shift, is the modulation frequency and is the speed of light.

Figure 2 :

 2 Figure 2: Simplified schematic of the resonant capacitive discharge driver topology.

1 T 2 (

 12 c) Current-mode driver in shunt configuration.

Figure 3 :

 3 Figure 3: Simplified schematic of the rectangular pulse driver topologies.

Figure 4 :

 4 Figure 4: Simplified schematic of the voltage-mode driver topology in series configuration.

Figure 5 :

 5 Figure 5: Simplified schematic of the current-mode driver topology in shunt configuration.

Figure 6 :

 6 Figure 6: Simplified block diagram of the two driver ICs.The current-control loops are inherent to the DC/DC converters.

Figure 7 :

 7 Figure 7: Physical layout of the HFS block.

Figure 8 :Figure 9 :

 89 Figure 8: Simulation transient waveforms for the voltage-mode driver: gate-source voltage of transistor T SW (top), laser diode current I LASER (middle), switching node voltage V SW (bottom, blue) and output voltage V OUT (bottom, green) for a 3A peak current with a 2.5ns pulse width at 200MHz under typical conditions.

  Test board with LDD prototype coupled to photodetector (viewed from inside the box, a zoom presents the IC, VCSEL diode and passive components).

Figure 10 :

 10 Figure 10: Test bench for performing electrical and optical measurements. Due to laser safety standards, measurements have been performed in a laser safety box protected with an interlock.

Figure 11 :

 11 Figure 11: Measured bandgap voltage vs. desired peak current for both drivers and at various frequencies under typical conditions.

Figure 12 :

 12 Figure12: Transient measurement waveforms for the voltagemode driver for a 2.5W peak optical power with a 2.5ns pulse width at 200MHz under typical conditions. The laser pulse shape is normalized on y-axis according to its maximum value. Important: electrical and optical waveforms are not in phase.

Table 1

 1 Main electro-optical characteristics of the VCSEL diode at 25°C.

	Parameter	Value
	Wavelength	940nm
	Peak optical power (under pulsed conditions)	2.4W
	Threshold current	300mA
	Series resistance	400mΩ
	Nominal current (under pulsed conditions)	3A
	Forward voltage	2.7V
	Slope efficiency	90%
	Power Conversion Efficiency	28%

Table 2

 2 Post-layout simulation results for a 3A peak current with a 2.5ns pulse width at 200MHz under typical conditions.

	Parameter	Value
	Driver	Voltage-mode	Current-mode
	Pulse width (50%)	2.6ns	2.5ns
	Rising time (10%-90%)	1.9ns	1.0ns
	Falling time (90%-10%)	900ps	2.2ns
	Peak current	3.5A	3.7A
	Average current	1.5A	1.8A
	Output voltage	5.0V	-
	Voltage spike at V SW	7.8V	7.9V
	Output power	4.2W	4.9W
	Driver input power	6.8W	8.2W
	Dissipated power in IC	2.6W	2.8W
	Electrical efficiency	62%	60%

Table 3

 3 Results summary for both drivers for a 2.5W peak optical power with a 2.5ns pulse width at 200MHz under typical conditions.

	Parameter	Value
	Driver	Voltage-mode	Current-mode
	Peak optical power	2.47W	2.51W
	Peak current	3.05A	3.09A
	Output power	4.50W	4.49W
	Driver input power	7.71W	7.25W
	Electrical efficiency	58%	62%
	Pulse width at 50%	2.6ns	2.5ns
	Rising time at 10%-90%	1.6ns	460ps
	Falling time at 90%-10%	410ps	1.2ns
	the output voltage which are mostly due to ESL of decou-
	pling capacitors. Nevertheless, a voltage spike of around 7V
	has been observed on the switching node for both prototypes
	suggesting that the clamping diode is less efficient than ex-
	pected.

Acknowledgments

The authors would like to thank STMicroelectronics for providing the 130nm CMOS technology and supporting the fabrication of the integrated circuit prototypes as well as the acquisition of the measurement equipment.

The authors also invite to review the role of scientific researchers and engineers by fully re-considering environmental, social and ethical requirements in the development of a concept, a product or a service.


Delphine Winstel
email: delphine.winstel@u-bordeaux.fr

Delphine Bahammou
email: delphine.bahammou@u-bordeaux.fr

Warren Albertin
email: warren.albertin@u-bordeaux.fr

Pierre Waffo-Téguo
email: pierre.waffo-teguo@u-bordeaux.fr

Axel Marchal
email: axel.marchal@u-bordeaux.fr

Untargeted LC-HRMS profiling followed by targeted fractionation to discover new taste-active compounds in spirits

Keywords: untargeted approach, metabolomic, LC-HRMS, taste-active compounds, sweetness, quantitation

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

Introduction

Beyond satisfying physiological needs, the consumption of food and beverages can provide pleasure to consumers thanks to their smell, taste and texture. For instance, enjoying a glass of spirits can be one of the delights of life, especially when it is old. It is well established that the value and the quality of cognac, whisky or rum significantly increase with their age.

From a molecular point of view, such sensory enjoyment depends on a wide range of volatile and non-volatile compounds. Deciphering the molecules responsible for the taste of natural food and beverages has been a great challenge for scientists not only in their quest for knowledge but also for commercial reasons. A better understanding of the origin of taste in food products can result in the more accurate monitoring of their production and consequently better preservation of their identity and diversity.

For several years, the search for taste-active compounds has been based on the bioguided purifications commonly used in pharmacognosy [START_REF] Peters | Identification of anabolic steroids and derivatives using bioassay-guided fractionation, UHPLC/TOFMS analysis and accurate mass database 29 searching[END_REF]. In such studies, sensory analysis was used to perform the fractionation of various foods and drinks [START_REF] Frank | Bioresponse-guided decomposition of roast coffee beverage and identification of key bitter taste compounds[END_REF][START_REF] Hufnagel | Orosensory-Directed Identification of Astringent Mouthfeel and Bitter-Tasting Compounds in Red Wine[END_REF][START_REF] Spreng | Activity-Guided Identification of in Vitro Antioxidants in Beer[END_REF]. For over 10 years, this approach has been implemented in our laboratory to isolate taste-active compounds from oak wood extracts, seeds and wine [START_REF] Cretin | Taste-guided isolation of sweet-tasting compounds from grape seeds, structural elucidation and identification in wines[END_REF][START_REF] Marchal | Identification of New Natural Sweet Compounds in Wine Using Centrifugal Partition Chromatography-Gustatometry and Fourier Transform Mass Spectrometry[END_REF][START_REF] Sindt | Taste-Guided Isolation of Bitter Lignans from Quercus petraea and Their Identification in Wine[END_REF].

The success of this inductive approach is based on the choice of raw extract, the reliability of the sensory test, and on the access to various complementary analytical techniques. However, wine and spirits are complex matrices containing thousands of compounds, so separation efficiency combined with potential sensory interactions during the tasting of fractions are limitations of this targeted strategy.

Untargeted analysis has been the subject of numerous developments for studying natural products [START_REF] Hoffmann | Improving Natural Products Identification through Targeted LC-MS/MS in an Untargeted Secondary Metabolomics Workflow[END_REF]. Metabolomics is an emerging and developing field for the chemistry of natural substances. High-performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) are the main techniques used to investigate non-volatile molecules in these approaches [START_REF] Wolfender | Innovative omicsbased approaches for prioritisation and targeted isolation of natural products -new strategies for drug discovery[END_REF]. More particularly, high-resolution mass spectrometry (HRMS) is very efficient thanks to its sensitivity, its resolution and its wide dynamic range. Since these techniques generate a large amount of data related to all the compounds detected in a sample, bioinformatics tools are required [START_REF] Blaženović | Software Tools and Approaches for Compound Identification of LC-MS/MS Data in Metabolomics[END_REF]. The objective is to highlight the significant information contained in the data collected.

Untargeted differential analysis has already been applied in enology to grapes [START_REF] Son | Metabolomic Studies on Geographical Grapes and Their 30 Wines Using 1H NMR Analysis Coupled with Multivariate Statistics[END_REF], oak wood extracts [START_REF] Gougeon | Expressing Forest Origins in the Chemical Composition of Cooperage Oak Woods and Corresponding Wines by Using FTICR-MS[END_REF], wines [START_REF] Gil | Discrimination of rosé wines using shotgun metabolomics with a genetic algorithm[END_REF] and spirits [START_REF] Kew | Chemical Diversity and Complexity of Scotch Whisky as Revealed by High-Resolution Mass Spectrometry[END_REF]. While high-performance liquid chromatography has proven to be an effective method for the analysis of spirits and has contributed to a better understanding of their chemical composition [START_REF] Collins | Profiling of nonvolatiles in whiskeys using ultra high pressure liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS)[END_REF][START_REF] Kew | Chemical Diversity and Complexity of Scotch Whisky as Revealed by High-Resolution Mass Spectrometry[END_REF], metabolomic approaches have recently shown great potential for their study [START_REF] Garcia | Whisky analysis by electrospray ionization-Fourier transform mass spectrometry[END_REF][START_REF] Stupak | A novel approach to assess the quality and authenticity of Scotch Whisky based on gas chromatography coupled to high resolution mass spectrometry[END_REF]. Most of these analyses were carried out to discriminate one or several cooperage parameters in order to authenticate spirits or simply to study the matrix [START_REF] Møller | Electrospray ionization mass spectrometry fingerprinting of whisky: Immediate proof of origin and authenticity[END_REF][START_REF] Roullier-Gall | Influence of regionality and maturation time on the chemical fingerprint of whisky[END_REF]. While global analysis can produce conclusions only of a descriptive nature, its use coupled with tools allowing screening appears promising for the discovery of new active substances [START_REF] Cabral | Targeted Isolation of Indolopyridoquinazoline Alkaloids from Conchocarpus fontanesianus Based on Molecular Networks[END_REF][START_REF] Nothias | Bioactivity-Based Molecular Networking for the Discovery of Drug Leads in Natural Product Bioassay-Guided Fractionation[END_REF]. Moreover, an exclusively untargeted metabolomic approach has limitations since it does not take sensory aspects into consideration.

Cognac is produced by distilling fermented grape juices mainly of the Ugni-blanc variety (Vitis vinifera Trebbiano), then leaving the spirit to age in oak barrels for a period of at least two years to several decades. This period of maturation is particularly important for spirits since they are devoid of non-volatile molecules at the beginning of aging. With aging the product becomes unique, gaining in flavor and revealing its color [START_REF] Piggott | Flavour of Distilled Beverages: Origin and Development[END_REF]. Thus, aging in oak barrels plays a key role in the sensory perception of spirits [START_REF] Cantagrel | Élaboration et Connaissance des Spiritueux: Recherche de la qualité, tradition & innovation[END_REF]. Several phenomena can induce physicochemical and sensory modifications [START_REF] Piggott | Flavour of Distilled Beverages: Origin and Development[END_REF]. In particular, extractables are released from oak wood and insoluble macromolecular compounds such as lignin are degraded, followed by the release of the degradation products of lignin. In addition, spirits undergo many chemical reactions and some extractables lead to the formation of new chemical species. A recent study showed that the diversity of compounds in aged spirits is greater than in the wood itself [START_REF] Roullier-Gall | Usage of FT-ICR-MS Metabolomics for Characterizing the Chemical Signatures of Barrel-Aged Whisky[END_REF]. This finding suggests that in spirits aged in barrels, various compounds natively present in the wood co-exist with molecules newly formed during aging. Several studies have focused on the composition of non-volatiles present in spirits but only a few have focused on taste-active compounds belonging in particular to the family of lignans, triterpenes and coumarins [START_REF] Gammacurta | Triterpenoids from Quercus petraea: Identification in Wines and Spirits and Sensory Assessment[END_REF][START_REF] Gammacurta | Isolation of Taste-Active Triterpenoids from Quercus robur: Sensory Assessment and Identification in Wines and Spirit[END_REF][START_REF] Winstel | Role of Oak Coumarins in the Taste of Wines and Spirits: Identification, Quantitation, and Sensory Contribution through Perceptive Interactions[END_REF]Winstel & Marchal, 2019). Better knowledge of the composition of spirits would help to explain why the taste of cognac improves with aging in barrels. The combination of non-targeted and targeted approaches would throw light on this issue.

The present study attempted to provide a molecular explanation for the increase in sweetness observed in old spirits and, in doing so, to propose a new methodology for the discovery of taste-active compounds. For this purpose, untargeted metabolomic profiling by HRMS was carried out on several "eau-de-vie" of Cognac of different vintages. Then, statistical analyses (principal component analysis (PCA), k-means clustering and Pearson correlation) were performed to assess the overall structure of the metabolomic data and to select compounds of interest. Their combined use with separative techniques such as liquidliquid extraction, centrifugal partition chromatography (CPC) and preparative-HPLC aimed to isolate new taste-active compounds that can be further identified, characterized for their sensory properties and quantitated in spirits.

Material and methods

Chemicals

Ultrapure water (Milli-Q purification system, Millipore, France) and HPLC-grade solvent (acetonitrile, ethanol, ethyl acetate, heptane, methanol and 1-butanol from VWR International, Pessac, France) were used for sample preparation and compound purification. Methyl tertbutyl ether (MTBE) was purchased from Scharlab S.L (Barcelona, Spain). HPLC-HRMS chromatographic separations were performed with LC-MS grade acetonitrile, deionized ultrapure water and formic acid (Optima, Fisher Chemical, Illkirch, France).

Samples

For the metabolomic approach, a set of spirits (Table S1, Supplementary Data), supplied by Rémy-Martin, was used. Ten different vintages (1970, 1973, 1990, 1993, 1995, 2000, 2005, 2008, 2010 and 2015) of "eau-de-vie" of cognac from the same distillery and having undergone similar aging conditions were tested. The samples were not commercial cognac but "eau-de-vie" still in barrels. They were matured in used barrels (350-L coarse-grain oak barrels). For each year, a sample was collected from five different barrels (except for 1970 and 1973 for which only four replicates were available).

An oak wood extract (25 g) was macerated in 250 mL of a hydroethanolic solution (50:50, v/v) at room temperature for three days in order to perform targeted LC-HRMS screening.

The spirit sample C-32 (Table S1, Supplementary Data), and a white wine aged 8 months in oak barrels (Rhône Valley, 2010, 14% vol. alc.) were also used.

Oak wood used to isolate targeted compounds was supplied by the cooperage company Seguin-Moreau (Merpins, France). It was sampled in April 2017 from a batch of staves used to make barrels. The botanical species was assigned to Quercus petraea according to the method described by [START_REF] Marchal | New Approach for Differentiating Sessile and Pedunculate Oak: Development of a LC-HRMS Method To Quantitate Triterpenoids in Wood[END_REF]. The staves were air-dried for two years according to the cooperage process. They were then reduced to sawdust by the barrel manufacturer (Seguin Moreau, Merpins, France).

For quantitative analysis, Alc-diHDDC was assayed in the same spirits as those described above for the metabolomic approach. The second set of spirits consisted of 28 commercial spirits aged in oak wood (with 12 cognacs, 5 whiskies, 4 grape brandies, 4 bourbons and 3 rums). The spirit samples were reduced to approx. 8% alcohol and then filtered at 0.45 µm.

The dilution factor applied was taken into account when calculating the final concentration.

HPLC Analysis

Metabolomic approach

For the non-targeted profiling approach, the HPLC appliance consisted of a Waters Acquity I-Class UPLC system (Waters, Guyancourt, France). For liquid chromatography separation, a C18 column was used as the stationary phase (BEH C18, 2.1 mm × 100 mm, 1.7 μm particle size; Waters). The mobile phases were water containing 0.1% formic acid (Eluent A) and acetonitrile with 0.1% formic acid (Eluent B). The flow rate was set at 400 μL/min and the injection volume was 2 μL. Eluent B varied as follows: 0 min, 5% (curve: initial); 0.5 min, 5% (curve: 6); 10.5 min, 100% (curve: 6); 14 min, 0% (curve: 11); 16 min, 0% (curve: 11).

Targeted fractionation and quantitation

For the screening and quantitative analysis, the HPLC appliance consisted of an HTC PAL autosampler (CTC Analytics AG, Zwingen, Switzerland) and an Accela U-HPLC system with quaternary pumps.

Liquid chromatography separation was performed on a C18 column (Hypersil Gold 2.1 mm × 100 mm, 1.9 μm particle size; Thermo Fisher Scientific), with water containing 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) as mobile phases for screening analysis.

The flow rate was 600 μL/min and eluent B varied as follows: 0 min, 10%; 1.0 min, 10%; 5.0 min, 50%; 5.3 min, 98%; 6.0 min, 98%; 6.15 min, 10%; 7 min, 10%. For the quantitative analysis, the column, the flow rate and the solvents used were the same as for screening analysis. However, eluent B varied as follows: 0 min, 5%; 1.6 min, 5%; 6.3 min, 50%; 7.0 min, 98%; 8.0 min, 98%; 8.1 min, 5%; 9.3 min, 5%. The injection volume was 5 μL for each method.

HRMS

For the metabolomic approach, a Q-Exactive Plus mass spectrometer equipped with a heated electrospray ionization (HESI-II) probe (both from Thermo Fisher Scientific, San Jose, CA) was used. For the screening and quantitative analysis, an Exactive Orbitrap mass spectrometer was equipped with a heated electrospray ionization (HESI-II) probe (both from Thermo Fisher Scientific). Each mass analyzer was calibrated with Pierce ® ESI Positive Ion Calibration solutions each week (Thermo Fisher Scientific).

The spectrometric and ionization parameters were not similar for the three types of analysis and are summarized in Table 1. Optimization of voltages, gas values, and temperatures applied for ion transfer and ionization was performed in positive mode.

For the non-targeted profiling approach, the data-dependent MS 2 events were performed on the four most intense ions detected by full-scan MS.

For quantitative and screening analysis, detection of Alc-diHDDC (compound A) was based on the theoretical exact mass of the most intense ion, the fragment ion at m/z 331.1531, and its retention time at 5.57 min. Peak areas were determined by automatic integration of extracted ion chromatograms built in a 3-ppm window around the exact mass of the C19H23O5 + ion.

Quan and Qual Browser applications of Xcalibur (version 3.0, Thermo Fisher Scientific) were used to process all the data.

MS Data Processing

After analyzing the spirit samples on the Q-Exactive Plus, Thermo RAW files were exported to the open source software package MZmine 2 (2.38 version) for data processing [START_REF] Pluskal | MZmine 2: Modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data[END_REF]. The protocol implemented includes five major steps: peak detection (mass detection), chromatogram builder, peak deconvolution, isotope suppression and peak list alignment. Each chromatogram was deconvoluted into individual chromatographic peaks with the "baseline cut-off" algorithm, which recognizes each chromatographic peak that has an intensity above a given minimum level (2.10 3 ) and spans over a given minimum time range (0.01-1.00 min). Chromatograms were deisotoped using the isotopic peaks grouper algorithm with a m/z tolerance of 10 ppm and a RT tolerance of 0.1 min. Peak alignment was performed using the Join aligner method. The last step was a "gap-filling" procedure, which reduces the number of missing values. All the parameters are described in Supplementary Data (Table S2). The generated peak lists were exported as comma-separated values (CSV) files to Excel 2016.

Statistical analyses

All the statistical analyses were carried out using the open-source software R Statistical (Foundation for Statistical Computing, Vienna, Austria). Results were interpreted by one-way analysis of variance (ANOVA), using vintage as factor. The values were previously transformed into logarithms in order to improve the homogeneity of the variances, a condition for applying the ANOVA. The normal distribution of the data was tested using the Shapiro-Wilk test. PCA was performed on normally distributed data. K-means clustering was also established for data classification (package ClassDiscovery). Pearson correlations were then carried out (α < 0.05, correlation coefficient r > 0.8) and allowed the creation of groups of compounds with a similar evolution depending on the vintages. The Pearson coefficient is an index reflecting a linear relationship between two continuous variables. A positive correlation indicates that the two variables vary together in the same direction. It was calculated according to the following formula:

= ∑ - - ∑ - ∑ -²
All p-values were corrected for multiple testing using the Benjamini-Hochberg correction (False Discovery Rate).

Purification protocol and structural elucidation 2.7.1. Extraction of oak wood

The oak wood material (600 g) was macerated in 6 L of H2O/EtOH solution (50:50; v/v) at room temperature for 2 weeks, under an inert atmosphere and protected from light. Filtration (0.45 μm) was used to remove wood sawdust and particles. Then, it was pressed for 15 minutes at a pressure of 6 bar using a pneumatic press. The solution containing soluble wood compounds (5.5 L) was concentrated in vacuo by evaporation of ethanol and, partly, water.

The aqueous extract (900 mL) was washed twice with 450 mL of n-heptane. This aqueous layer was then extracted with MTBE (6 × 500 mL). The combined organic layers were evaporated in vacuo, suspended in water, and freeze-dried twice to obtain brownish powders of MTBE (1.8 g) and aqueous (20.1 g) pre-purified extracts. They were stored under air-and light-protective conditions.

Centrifugal partition chromatography fractionation

CPC was performed on a Spot prep II LC coupled with an SCPC-100+1000 (Armen Instrument, Saint-Ave , France), both controlled by Armen Glider Prep V5.0 software. A 100-mL rotor (11 discs, 90 twin cells of 0.101 mL per disc) was used to fractionate samples weighing less than 1 g and a 1-L rotor (21 discs, 72 twin cells of 0.555 mL per disc) was employed for samples weighing more than 1 g. The solvent was pumped into the column by a 4-way quaternary high-pressure gradient pump. The samples were introduced into the CPC column via an automatic high-pressure injection valve. All the experiments were conducted at room temperature, with UV detection at 254 and 280 nm.

The choice of an appropriate biphasic system of solvents was based on the study of the partition of extract compounds in both phases according to the procedure described by [START_REF] Marchal | Identification of New Natural Sweet Compounds in Wine Using Centrifugal Partition Chromatography-Gustatometry and Fourier Transform Mass Spectrometry[END_REF]. The partition coefficient, , was calculated as the ratio of the solute area in each phase. On this basis, various systems were tested, and the MTBE extract was fractionated using the Arizona solvent systems H (n-heptane/EtOAc/MeOH/H2O, 1:3:1:3 Ten fractions F-I to F-X were constituted on the basis of their similar chromatographic profile, after being combined, evaporated in vacuo, suspended in water and freeze-dried.

v/v/v/v)
The CPC fraction F-II (333.8 mg) was rich in targeted compound A. A second CPC experiment was carried out on this fraction using the Arizona-K system (nheptane/EtOAc/MeOH/H2O, 1:2:1:2 v/v/v/v). It was performed in ascending mode at 2300 rpm with a flow rate of 5 mL/min for 70 min for the elution phase and 10 mL/min for 30 min for the extrusion. Seven fractions were obtained (F-II_1 -F-II_7). S3, Supplementary Data.

Preparative liquid chromatography

NMR experiments

NMR experiments were conducted on a Bruker UltraShield ® System Avance 600 NMR, spectrometer ( 1 H at 600.27 MHz and 13 C at 150.95 MHz) equipped with a 5 mm TXI probe.

All 1D (proton) and 2D ( 1 H-1 H COSY, 1 H-1 H ROESY, 1 H-13 C HSQC, and 1 H-13 C HMBC)

spectra were acquired at 300 K in methanol-d4, which gave as reference the solvent signal ( 1 H δ 3.31; 13 C δ 49.00). Data analysis was performed with the NMRnotebook software (NMRTEC, Illkirch-Graffenstaden, France).

Polarimetry

Optical rotation was measured with a JASCO P-2000 polarimeter. The sodium emission wavelength was set at 589 nm and the temperature at 25 °C. The pure compound was dissolved in MeOH, and the final value resulted from a mean of 10 measurements of 10 s each.

Sensory characterization

Gustatory analysis was performed in a dedicated room, under normal daylight, and at room temperature (around 20°C). Pure compounds were tasted by five experts in winetasting, at 2 mg/L in water (eau de source de Montagne, Laqueuille, France), as well as in a non-oaked "eau-de-vie" adjusted to 40% v/v. Experts described the gustatory perception (bitterness, sourness, sweetness) of the targeted compound using the vocabulary of winetasting and were asked to evaluate the intensity on a scale from 0 (not detectable) to 5 (strongly detectable).

The tasters were informed of the nature and risks of the present study and were asked to give their consent to participate in the sensory analyses. Even though the compound was purified from commercial oak wood used for cooperage and observed in spirits, the panelists were advised not to swallow but to spit out the samples after tasting.

Quantitation of purified compound in spirits 2.9.1. Preparation of calibration solution

A stock solution of Alc-diHDDC (1 g/L) was prepared in ethanol. Successive dilutions of this solution were performed with ultrapure water to provide calibration samples (10 mg/L, 5 µg/L and 1 µg/L).

Validation method for quantitating Alc-diHDDC

The method for quantitating Alc-diHDDC in spirits was validated by studying linearity, intraday repeatability, sensitivity, trueness and specificity. The sensitivity of the LC-HRMS method was determined according to the approach described by De Paepe et al. ( 2013). The limit of detection (LOD) of a molecule is defined as the lowest concentration at which a reproducible and reliable signal is observed. Moreover, it had to be different from a blank performed under the same conditions. The five lowest calibration points (1, 2, 5, 10 and 20 µg/L) were injected five times. Limit of quantitation (LOQ) corresponded to the lowest concentration of the molecule that can be quantitatively determined by the method, with an accuracy (recovery of back-calculated concentrations) higher than, e.g., 90% and a precision lower than, e.g., 10%. A calibration curve was created by plotting the areas for each concentration level versus the nominal concentration. Linearity was assessed by deviations of each back-calculated standard concentration from the nominal value and by correlation coefficient (R²). Three intermediate calibration solutions (100 µg/L, 1 mg/L and 10 mg/L) were injected into five replicates to define intraday precision. The calculation of relative standard deviation (RSD%) was carried out. Trueness was verified by calculating the recovery ratio (between expected and measured areas) from two analyzed samples of "eau-de-vie" (EDV-1; EDV-2). They were spiked with calibration solution (addition of 10 µg/L, 200 µg/L and 10 mg/L) of Alc-diHDDC. Injections of the same two samples (10 µg/L and 10 mg/L) for five consecutive days were performed to estimate interday repeatability. Specificity was evaluated by assessing retention time repeatability and mass accuracy.

Results and discussion

Metabolomic Fingerprinting of cognac spirits

An untargeted analysis by HRMS was performed on a series of "eau-de-vie" of cognac of 10 different vintages from 2015 to 1970. The 48 samples, which were collected in the same distillery and aged in similar conditions, were not commercial cognac but "eau-de-vie" still aging in barrels (Table S1, Supplementary Data). For each vintage, a sample was collected from four or five different casks to limit variations due to cask effect.

The main objective of this untargeted profiling was to observe potential differences in composition between the young and the old vintages of "eau-de-vie" of cognac by a global analysis. To this end, the chromatographic conditions were set up in order to optimize the separation of the compounds present in these spirits. The spectrometric parameters were also adapted to enhance the sensitivity of a maximum of non-volatile molecules. After the HPLC-HRMS analysis, the data were processed according to the metabolomic strategies implemented for the research of natural products [START_REF] Wolfender | Innovative omicsbased approaches for prioritisation and targeted isolation of natural products -new strategies for drug discovery[END_REF], with the use of MZmine 2 software. Thanks to these treatments, a peak list of 47,243 positive ions was obtained between m/z 100 and 1500, then filtered into a peak list of 1,293 ions having an associated data-dependent MS 2 spectrum. These results revealed the chemical diversity and complexity of cognac, which is consistent with previous studies on different spirits [START_REF] Kew | Chemical Diversity and Complexity of Scotch Whisky as Revealed by High-Resolution Mass Spectrometry[END_REF][START_REF] Roullier-Gall | Influence of regionality and maturation time on the chemical fingerprint of whisky[END_REF]. The rest of this work was carried out on this set of ions.

It is important to note that the data reduction essential for statistical analyses is also likely to cause data loss, which could constitute a major limitation to this type of approach.

A metabolomic study sometimes involves a differential analysis of fingerprints, with semiquantitative expression of the results [START_REF] Boudah | Chapter Four-Liquid Chromatography Coupled to Mass Spectrometry-Based Metabolomics and the Concept of Biomarker[END_REF]. Here, a PCA of the data was carried out using the peak areas of the 1,293 positive ions highlighted by the ANOVA. The two factorial axes of the positive mode explained 79.7% of the data set (Figure 1,A). The vintage effect was clearly significant on the first axis (that explained 71.5% of the total variation of the data set). Moreover, ANOVA showed that the vintage effect was significant (p < 0.05) for 1,036 compounds out of 1,293 (80%). K-means clustering followed by Pearson correlations allowed us to divide the compounds into different groups showing similar trend to evolve according to the vintages. Of the 1,036 compounds detected in positive mode and significantly influenced by the vintage, 854 were assigned to a group among the five created (Figure 1,B). Groups 2 and 3 represented 82% of the compounds whose concentrations were significantly influenced by the vintage, which were more abundant in older vintages. Group 5 was composed of 80 compounds whose contents increased during 23 years of aging and then slowly decreased. Group 1 contained 10 compounds present in higher concentration in young "eau-de-vie" of cognac and then less abundant during aging. Group 4 was composed of 60 molecules whose contents were relatively constant over the first 30 years of aging and decreased significantly thereafter. The group evolutions (Figure 1, B) suggested that most of the compounds (703/854) were present at higher concentrations in old spirits and that there were more compounds in old vintages. This observation was consistent with the dry extract contents obtained for the 10 cognac vintages: higher in the old vintages than in the young ones (Table S1, supplementary data).

The creation of statistical groups revealed the presence of a wide variety of compounds in these "eau-de-vie" of cognac. For most of them, the concentrations were higher in aged spirits, while the opposite was observed for less than 20% of the compounds. Two hypotheses could explain these observations: either the compounds were released from wood to spirits continuously during aging, or the compounds were neoformed by chemical reactions in the spirits.

Untargeted metabolomic analysis to select and purify molecules of interest

In general, the last step in a metabolomic analysis aims at tentatively or formally identifying the compounds associated with the detected signals. This step is the most hazardous because ESI ionization often produces more than one ion for a given metabolite. These ions can correspond to fragments produced in the source, pseudo-dimers, adducts or their isotopic derivatives that have passed through the filters of the treatment. Moreover, identification of the compounds generally requires the combined use of several analytical techniques such as mass spectrometry, NMR, UV or IR. During an LC-MS approach, formal identification is generally based on the similarity of the chromatographic retention time, the exact mass and the MS n spectra between the studied compound and a standard or a compound present in spectral libraries. If some of these criteria are not validated, the characterization of the compound is not complete and the identification remains putative. Therefore, this tool cannot lead to the unambiguous identification of a new compound.

The identification of all the metabolites present in these "eau-de-vie" of cognac by dereplication was not undertaken because it is time-consuming and only a few non-volatile compounds are known to this day. Instead, the peak list obtained by the MZmine analysis and the data from the statistical analysis were used to target new natural products. We focused particularly on compounds that were formed during the aging of the spirits, since they were more likely to contribute to taste modifications and tried to purify them.

First, the creation of a table enabled us to classify the molecules according to their intensity in the spirits from 1970 to 2015 (data not shown). Subsequently, LC-HRMS screening of these molecules in this matrix and in oak wood extracts was investigated. Thanks to its mass measurement accuracy and separative performance, LC-HRMS is a powerful technique to screen complex matrices. The compounds of interest were selected according to two criteria: a significant abundance of the targeted compound and a strong increase in concentrations in old vintages. Despite the large number of chromatographic peaks, a compound combining the previously defined criteria was targeted for the rest of the study:

compound A with a nominal mass of 331. It was indeed classified among the compounds with the greatest intensity difference between the spirits of 1970 and 2015, with an increase of 3,142%, and it is one of the 475 compounds present in Group 2 (Figure 1, B). By LC-HRMS screening, compound A was also detected in an oak wood extract (Figure S4, Supplementary Data). Extracted ion chromatograms (XICs) were built by targeting the positive ion at m/z 331.1531 within a window of 5 ppm around its theoretical m/z. In addition, the HRMS/MS spectra revealed the same fragment signals in both matrices with retention times of 4.02 minutes (Figure S4, supplementary data). Therefore, compound A is present natively in wood.

Its content in aged spirits may therefore be explained by its continuous release during maturation in barrels.

For reasons of availability of materials, its purification protocol was carried out using extracts of oak wood. Its sensory properties as well as its structural characterization were established.

Isolation and identification of compound A in spirits

Extraction and purification of compound A from oak wood

To reproduce cellar conditions, a hydroethanolic solution was used to perform the solid-liquid extraction of wood sawdust of Quercus petraea. Thus, the extracted molecules present in this solution had to be the same as those released in an "eau-de-vie" aged in barrels. After removal of ethanol, the first step consisted of sequential liquid-liquid extractions using MTBE to remove compound A from the aqueous solution. The chemical complexity of the enriched MTBE extract required a further fractionation by CPC. Systems containing relatively apolar solvents from the Arizona range were tested for this fractionation. A gradient from Arizona-H system to Arizona-G allowed the best partition of the MTBE extract between the two phases.

spectrum also presented an ion at m/z 383.1463 corresponding to a sodium adduct [M+Na] + (C20H24O6Na + ) (Figure S5, Supplementary Data).

By comparing the data obtained in LC-HRMS, the interpretation of 1 H and 13 C NMR spectra (Figure S6, Supplementary data) and the data from the literature, compound A was identified as dihydrodehydrodiconiferyl alcohol (Alc-diHDDC). The determination of its structure from NMR data is not detailed here since it has already been described in the literature [START_REF] Hanawa | Heartwood constituents of Betula Maximowicziana[END_REF]. However, it was necessary to specify its relative configuration. It has been demonstrated that the coupling constant between H-7 and H-8

(equal to 6.5 Hz here) cannot unambiguously distinguish the cis/trans diastereoisomers with a skeleton of this type [START_REF] García-Muñoz | Synthesis of dihydrodehydrodiconiferyl alcohol and derivatives through intramolecular C-H insertion[END_REF]. The interpretation of the 1 H-1 H ROESY spectrum showed a correlation between H-7 (δH 5.50, d) and H-9 (δH 3.75, m). This observation indicates that the protons H-7 and H-8 were in the trans position, as detailed in the literature [START_REF] García-Muñoz | Synthesis of dihydrodehydrodiconiferyl alcohol and derivatives through intramolecular C-H insertion[END_REF] (Figure 2).

This neolignan was highlighted for the first time in 1970 in Silybum marianum [START_REF] Suga | Phenolic natural products. XIII. Isolation and determination of the constitution of an optically active dehydrodiconiferyl alcohol from the seeds of the milk thistle Silybum marianum[END_REF] and has been identified in several natural species such as Pinus massoniana [START_REF] Suga | Phenolic natural products. XIII. Isolation and determination of the constitution of an optically active dehydrodiconiferyl alcohol from the seeds of the milk thistle Silybum marianum[END_REF], but never in the Quercus genus. It has also been found in maple syrup and maple sugar [START_REF] Liu | Isolation, Identification, and Biological Evaluation of Phenolic Compounds from a Traditional 28 North American Confectionery, Maple Sugar[END_REF] but no publication mentions its presence in spirits. Thus, this study allowed the first identification of Alc-diHDDC in oak wood and cognac. Moreover, a white wine aged in oak barrels was screened by LC-HRMS. The presence of Alc-diHDDC was also assessed in this matrix which appeared consistent with its release from oak wood (Figure S4, supplementary data).

Gustatory properties of targeted compound A

This study is the first to assess the gustatory properties of Alc-diHDDC. It was dissolved in water and in a non-oaked "eau-de-vie" at 2 mg/L, and the taste of each solution was characterized in comparison to the same water / "eau-de-vie" as a reference. QTT I was used as a sweetness standard since its sensory properties have already been characterized [START_REF] Marchal | Identification of New Natural Sweet Compounds in Wine Using Centrifugal Partition Chromatography-Gustatometry and Fourier Transform Mass Spectrometry[END_REF]. In water, Alc-diHDDC exhibited a sweet taste. On a 0-5 scale representing relative sweetness intensity assessed as a consensus between the five panelists, Alc-diHDDC scored 4/5 and QTT I was assessed as 2/5. Alc-diHDDC was also dissolved in non-oaked "eau-de-vie" to study its influence on the taste balance of spirits. The control "eau-de-vie" was scored 0/5 for sweetness and 5/5 for the perception of burning. As a reference, "eau-devie" spiked with QTT I (2 mg/L) was described as sweeter (4/5) and less burning (1/5). Alc-diHDDC also modified the taste of the "eau-de-vie" by decreasing the perception of burning

(2/5) and increasing that of sweetness (4/5).

The results suggested that Alc-diHDDC developed a sweet taste at 2 mg/L with an intensity close to that of QTT I, whose gustatory detection threshold is relatively low for nonvolatile compounds (i.e. 590 μg/L in wine [START_REF] Marchal | Recherches sur les bases moléculaires de la saveur sucrée des vins secs: Approches analytique et sensorielle[END_REF]), which is much lower than that of glucose, i.e., 4 g/L (Ribéreau- Gayon et al., 2006).

Quantitation of Alc-diHDDC in spirits by LC-HRMS

Method validation

Preliminary accuracy tests had shown that there was no significant matrix effect for this compound in spirits. Therefore, absolute quantitation was carried out by preparing calibration solutions of Alc-diHDDC in ultra-pure water. The limits of detection (LOD) and quantitation (LOQ) were established at 2 µg/L and 5 µg/L, respectively. For a range from 5 µg/L to 10 mg/L, a quadratic calibration curve (1/x statistical weight) with a good correlation coefficient (R² of 0.996) was obtained. Moreover, accuracy was established, since recovery of backcalculated concentrations was higher than 90% at each method calibration level. Intraday repeatability (RSD%) for each concentration was lower than 9.3%. Injections of two spirits spiked with stock solutions were also performed. Recovery ratios ranged from 85 to 95%, which remained in accordance with common specifications (Guidance for Industry, 2018).

Moreover, interday repeatability was < 10% at 10 µg/L and < 7% at 10 mg/L. Consequently, these results established the repeatability and the trueness of the method applied to spirits.

Analysis of the above samples revealed small variations in retention time (< 0.04 min) and a mass deviation lower than 0.3 ppm at various concentrations, guaranteeing the specificity of the method. All these results validated the LC-HRMS method to quantitate Alc-diHDDC in spirits (Table 2).

Application of method to quantitate Alc-diHDDC in spirits

Alc-diHDDC was quantitated in the samples of "eau-de-vie" of cognac used for untargeted metabolomic analysis (Table S7, Supplementary data). The concentrations presented in Figure 3 correspond to the mean values of the five replicates (Figure 3,A). The contents measured for Alc-diHDDC were higher in old vintages, until reaching an average concentration of 3.3 mg/L for the 1970 vintage. Thus, long aging in barrels seemed to promote the extraction of this compound. For 2008 and 2005 vintages, a high standard deviation was observed because of a high value in the series. For all the barrels, the aging conditions were similar, which suggests that these differences were due to heterogeneity in oak wood composition. Such variations have already been observed for volatile and non-volatile compounds (Marchal et al., 2016). These results were consistent with its relative quantitation (Figure S8, Supplementary Data) obtained from the untargeted metabolomic approach, for which the same trend was observed. More generally, these results confirmed the effectiveness and relevance of this approach.

Twenty-eight commercial spirits were analyzed to assess the range of Alc-diHDDC amounts in some cognacs, but also whiskies, rums, and other brandies, using the previously validated LC-HRMS method (Table S9, Supplementary Data). Error bars indicated the repeatability of the method. Alc-diHDDC was detected in all samples at concentrations ranging from 0.4 to 4.0 mg/L, with a mean value of 1.7 mg/L (Figure 3,B). The contents also varied according to the type of spirit, with higher values in rums and bourbons than in cognacs (except for sample C-12) and brandies. The higher Alc-diHDDC concentration in the C-12 sample was correlated with longer aging in barrels than for the other cognacs analyzed. The differences in concentration between the spirits could be due to the botanical origin of the wood used for aging. Indeed, cognacs and brandies are generally aged in French sessile or pedunculate oak barrels, while bourbons are aged in American oak barrels. The potential influence of aging conditions on Alc-diHDDC concentrations in spirits needs to be clarified in further studies.

Moreover, 9 of the 28 commercial spirits had concentrations of Alc-diHDDC greater than 2 mg/L (value used for tasting), which could suggest its sensory contribution to the sweetness of these spirits.

Conclusion

The aim of this work was to propose an efficient methodology for discovering new tasteactive compounds in oak wood and oaked spirits. A graphical summary of this approach is presented in Figure 4. An untargeted metabolomic profiling by HRMS was implemented on "eau-de-vie" of cognac from ten vintages. Firstly, a purely descriptive approach was undertaken. After statistical analysis, thousands of compounds were observed in these spirits.

Two categories of molecules emerged: those extracted from oak wood in spirits and those formed by chemical reactions in spirits. Among them, compound A (m/z 331) was chosen on the basis of two criteria including its high abundance in old vintages. After the development of a fractionation protocol using CPC and preparative HPLC, dihydrodehydrodiconiferyl alcohol (Alc-diHDDC) was identified and purified from oak wood extracts. Its presence in this matrix and in spirits has never been described until now. Sensory analysis revealed that Alc-diHDDC exhibits a sweet taste at 2 mg/L in water and in a non-oaked spirit. The development of a quantitative HPLC-HRMS method showed that some commercial spirits contained Alc-diHDDC at concentrations above this value. Even if these results established the sensory relevance of the newly identified compound, the determination of its gustatory detection threshold will establish its impact. Future work will clarify the cooperage parameters likely to affect its concentration in oak wood, wines and spirits.

These results demonstrate the relevance and the effectiveness of combining untargeted differential analysis to highlight the molecules significantly affected by barrel aging, and a targeted fractionation protocol to isolate them. This work offers promising perspectives for the search for new natural products and could also be coupled with the inductive approach, guided by the gustatometric analysis conventionally used in our laboratory.

  and then by setting up a gradient on the Arizona-G system (nheptane/EtOAc/MeOH/H2O, 1:4:1:4 v/v/v/v). Separation was performed by one CPC run of 1.8 g injection. Experiment was carried out in ascending mode at 1200 rpm with a flow rate of 30 mL/min for 145 min for the elution phase and 50 mL/min for 45 min for the extrusion. The Spot Prep fraction collector was set to 25 mL/min. Every 10 CPC tubes, an aliquot (200 μL) was taken, evaporated, dissolved in 1 mL of H2O/MeOH 95:5 and analyzed by LC-HRMS.

Figure 1 :

 1 Figure 1: (A) PCA representation based on 48 "eaux-de-vie" of cognac (10 vintages, from 2015 to 1970) and (B) Representation of different groups of compounds according to their evolution in same samples.

Figure 4 :

 4 Figure 4: Schematic diagram of new methodology used for discovering taste-active compounds.

  Quaternary Gradient Module, a 2489 UV/Visible detector and a 2424 ELSD detector (Waters, Guyancourt, France). Final purification of the targeted compound A, which was present in the CPC fraction F-II_5 (54.3 mg), was achieved by preparative HPLC using columns chosen after LC-HRMS tests. Separation was performed using a SunFire Prep C18 OBD (19 mm × 250 mm, 5 μm particle size; Waters) equipped with a SunFire preparative C18 guard cartridge (20 × 19 mm, 5 μm particle size; Waters). The mobile phase was a mixture of ultrapure water containing 0.1% formic acid (Eluent A) and acetonitrile with 0.1% formic acid (Eluent B).

	The flow rate was set to 20 mL/min. Eluent B varied as follows: 0 min, 20%; 7.4 min, 20%;
	37 min, 25%; 39 min, 98%; 44.2 min, 98%; 45.3 min, 20%; 52 min, 20%.
	Aliquots (20 mg) of CPC fractions were dissolved in 400 µL of H2O/MeOH 60:40, 0.45

μm-filtered and introduced manually into the system. UV detection was carried out at 280 nm and chromatographic peaks were collected manually just after the detector. The pure compound solution was evaporated in vacuo to remove acetonitrile and freeze-dried to obtain a white amorphous powder (3.1 mg). Dihydrodehydrodiconiferyl alcohol (A, Alc-diHDDC): white amorphous powder; [ ] -2.6 (c = 0.1, MeOH); HRMS m/z 361.1491 [M + H] + (C20H25O6 + , 1.3 ppm); 1 H NMR (methanol-d4, 600 MHz) and 13 C NMR (methanol-d4, 150 MHz), see Table

Table 2 :

 2 Validation parameters for HRMS quantitation of Alc-diHDDC in spirits.

	Parameters		Matrix -Spirits
	Sensitivity	LOD (µg/L) 2	LOQ (µg/L) 5
	Linearity and	Working range	R 2
	accuracy	5 µg/L-10 mg/L	0.996
	Specificity	tR variation 0.04 min	Mass accuracy 0.3 ppm
			Intraday repeatability
			10 µg/L	200 µg/L	10 mg/L
			9.3%	5.2%	5.3%
			Interday repeatability
	Repeatability and		10 µg/L	10 mg/L
	trueness		9.8%	6.7%
			Recovery
			100 µg/L	1 mg/L	10 mg/L
		EDV-1	85%	95%	94%
		EDV-2	87%	91%	94%

Acknowledgements

Delphine Winstel's grant is supported by Remy-Martin and Seguin-Moreau. Analytical techniques were funded by Biolaffort. The authors are also grateful to Dr. E. Frérot for performing LC-MS/MS experiments. We thank Dr. J.-C. Mathurin and Dr. L. Urruty for providing samples and Dr. Ray Cooke for proofreading the manuscript.

At the end of this experiment, the tubes were analyzed by LC-HRMS and pooled according to their composition to give fractions. After solvent evaporation and freeze-drying, 10 fractions (noted F-I to F-X) were obtained as powder in variable quantities. Fraction II (F-II) was rich in compound A so it was kept for the rest of the fractionation. It was still chemically complex and contained many compounds. Therefore, a second CPC experiment was performed.

Different solvent systems from the Arizona range were tested in order to choose the one allowing optimal fractionation. The Arizona-K system was selected and seven new fractions were obtained (noted F-II_1 to F-II_7).

After freeze-drying, fraction F-II_5, which was richer in compound A, was submitted to preparative HPLC with UV detection. A preliminary injection of 5 mg showed that the chromatograms presented a refined profile with only a few peaks detected both in ELSD and in UV at 280 nm. Thus, an appropriate gradient was chosen and the entire fraction F-II_5 was fractionated by successive injections. The peak corresponding to compound A was collected manually just after the UV detector to give 3.1 mg of a white amorphous powder after acetonitrile removal and freeze-drying.

Structural elucidation of compound A

The HRMS spectrum of compound A exhibited the most abundant positive ion at m/z 331.1531 (C19H23O5 + ). This corresponded to a fragment linked to the quasi-molecular ion [M+H] + , which was observed at a very low intensity whatever the ionization parameters. The protonated ion at m/z 361.1649 suggested an empirical formula of C20H24O6 (with an accuracy of 0.3 ppm). This molecule must be fragmented in the ionization source. Thus, the most detected species corresponded to a neutral loss of 29.9961 compared to the ion at m/z 361, characteristic of a methoxy group (CH2O). Moreover, the positive ion at m/z 313.1426 (C19H21O4 + ), could correspond to a dehydration in relation to the ion at m/z 331. The HRMS

Competing interests statement

The authors declare no competing financial interest.

Author contributions


Emilie Panicucci

MD Chiara Bruno

MD Victoria Ferrari

MD Laurent Suissa
email: suissa.laurent@free.fr

Recurrence of ischemic stroke on direct oral anticoagulant therapy in a patient with marantic endocarditis related to lung cancer

Keywords: marantic endocarditis, ischemic stroke, direct oral anticoagulant, lung cancer

Venous thromboembolism (VTE) and arterial thromboembolism (ATE) are frequent complications in cancer patients. For curative treatment of VTE in a neoplastic context, recent randomized controlled trials and meta-analyses suggest that direct oral anticoagulants (DOACs) can be an alternative to conventional treatment by low molecular weight heparins (LMWHs). On the other hand, the role of DOACs in the treatment of cancer-related ATE still remains unclear. Here, we report a case of stroke recurrence in a patient initially treated with DOACs that were started for cancer-related VTE. The failure of the DOACs, compared to LMWHs, to prevent the stroke recurrence led us to the infrequent final diagnosis of nonbacterial thrombotic endocarditis (NTBE, formerly known as marantic endocarditis). This case illustrates the association between ATE and VTE in a patient with marantic endocarditis related to lung cancer and recurrence of ischemic stroke on direct oral anticoagulant therapy.

Learning objectives:

• Arterial (ATE) and venous thromboembolism (VTE) are frequent complications in cancer patients • ATE and VTE can be responsible for ischemic stroke in neoplastic context

• VTE associated stroke in cancer patients does not exclude ATE etiology (marantic endocarditis)

• Direct oral anticoagulants fail to prevent ischemic stroke associated to marantic endocarditis

Introduction

For treatment of cancer patients with venous thromboembolism (VTE), guidelines recommend low molecular weight heparin (LMWHs). In recent years, meta-analysis based on indirect comparisons between LMWHs and direct oral anticoagulants (DOACs) suggests that DOACs have similar efficacy and safety to LMWHs for the treatment of cancer-associated thrombosis [START_REF] Posch | Treatment of venous thromboembolism in patients with cancer: A network meta-analysis comparing efficacy and safety of anticoagulants[END_REF]. However, cancer patients also have a high risk of developing arterial thrombosis with arterial thromboembolism (ATE), caused by both upregulation of coagulation and activation of platelets [START_REF] Varki | Trousseau's syndrome: multiple definitions and multiple mechanisms[END_REF]. The effect of DOACs on the treatment of cancer-related ATE still remains unclear.

Here, we report a case of ischemic stroke recurrences occurring with DOAC treatment administrated for documented VTE, leading to the discovery of marantic endocarditis in a neoplastic context.

Case report

A 64-year-old woman, with no cerebrovascular history, was admitted to the stroke unit for a sudden left hemiparesia. Her medical history recorded a partial thyroidectomy few months ago and active smoking. The patient consulted a clinic because of exertional dyspnea that had been evolving for approximately a month, associated with right calf pain occurred after a long walk a few days prior, leading to the diagnosis of right deep vein thrombosis (solear), complicated by a left distal pulmonary embolism (confirmed at the thoracic CT-scan). CT scan also showed pulmonary nodule requiring more explorations. The patient had normal vital signs (blood pressure 160/80, cardiac frequency 91/minute), including oxygen saturation (ambient air saturation 95%) and no sign of respiratory distress syndrome. Transthoracic echography conducted at this time showed no cardiopathy or signs of acute right heart failure (only a minor aortic insufficiency). Rivaroxaban 15 mg twice a day was prescribed immediately after the diagnosis.

A day after the initiation of rivaroxaban, she was admitted to our stroke unit because she presented sudden left hemiparesia with dysarthria. The NIHSS score was estimated to be 8, and she had no cardio-respiratory symptoms. A cerebral MRI was performed, showing multi-territorial ischemic strokes with different ages (Figure 1A). Indeed, recent ischemic stroke, as diagnosed by a hypersignal on the DWI sequence, were found in the right middle cerebral artery (MCA), right cerebral posterior and bilateral cerebellum territories, and the left superficial-profound MCA junction region. In this context of acute stroke, rivaroxaban was discontinued and treatment with therapeutic doses of intravenous continuous unfractionated heparin (UFH) was initiated immediately.

In the context of multi-territorial ischemic stroke, cardioembolic phenomenon was suspected, particularly paradoxical embolisms occurring during pulmonary embolism. Furthermore, supraaortic arteries were normal on the CT-scan, cardiac monitoring and biological tests showed no abnormality except for moderate elevation of cardiac troponin (722 ng/l) and D-dimer (35000 ng/ml initially, controlled at 7438 ng/ml after 10 days), related to pulmonary embolism. In particular, no inflammatory syndrome (C-reactive protein 14 mg/l) and thrombophilia tests were all negative (protein C, S, antithrombin III, antiphospholipid antibodies, Factor V Leiden mutation, factor II mutation) and disseminated intravascular coagulation syndrome was absent (Prothrombin Ratio 83%, platelets 216 x10 9 /l).

One week later, a neurological exam was stable, so UFH was substituted for DOAC (apixaban 5 mg twice a day). Two days later, the patient presented neurological aggravation with confusion and right hemianopsia. The NIHSS score was calculated to be 11. A new cerebral MRI was performed, showing new ischemic strokes (in DWI sequence), particularly in the left PCA-MCA junction region and the left posterior cerebral artery territory (Figure 1B). A treatment with LMWHs was introduced (enoxaparin 0.6 ml twice a day) and apixaban was discontinued.

In this context of recurrent ischemic strokes despite anticoagulation therapy and associated deep vein thrombosis and pulmonary embolism in a patient with no medical history and no thrombophilia at biological tests, with a lung nodule on first CT scan an underlying cancer was suspected. A thoracic-abdominal-pelvic CT-scan was then performed. The pulmonary nodule suspect of malignancy was found, associated to probable secondary liver and bone lesions. A CT scan was followed by a PET scan, which consolidated the hypothesis of cancer (Figure 2).

Bronchial fibroscopy with nodule biopsy was performed, leading to the diagnosis of primitive pulmonary adenocarcinoma by histological exam.

Given the context of lung cancer with recurrent ischemic strokes despite DOAC therapy, a new transthoracic echocardiography and transesophageal echography (Figure 3) was performed to identify a marantic endocarditis, responsible for ATE. These exams showed aortic valvular thickening with a doubt on aortic vegetation on transthoracic echocardiography, not find on transesophageal echography, but with severe aortic insufficiency, confirming the hypothesis of the endocarditis. No patent foramen ovale at bubble tests or interatrial septum aneurysm or interatrial thrombus was observed.

Therefore, in the absence of patent foramen ovale and given the delay between the pulmonary embolism and the onset of neurological symptoms, the hypothesis of paradoxical embolism was disproved. A diagnosis of marantic (non-infectious) endocarditis with ATE was established with transesophageal echography and results of complementary investigations (repeated hemocultures, viral serologies, anti-nuclear auto antibodies, rheumatoid factor) which were all negative.

The neurological exam was constant (NIHSS score of 8 and modified Rankin Scale of 5) and no recurrence of ischemic stroke was observed with enoxaparin, which was continued thereafter. The patient was later hospitalized in oncology for cancer treatment. Unfortunately, the pulmonary adenocarcinoma was already at an advanced stage, so the patient soon had significant cachexia associated with the neurological symptoms. She died a few days after the transfer to oncology, before the initiation of chemotherapy.

Discussion

This case report illustrates recurrence of ischemic stroke related to marantic endocarditis in a lung cancer context on direct oral anticoagulant therapy. Initially, ischemic strokes were imputed to a VTE phenomenon in neoplastic context. However, two stroke recurrences despite effective anticoagulation by DOACs and the absence of new ischemic lesions during heparin therapy lead us to consider other etiological mechanisms. We imputed an ATE phenomenon in the presence of a marantic endocarditis diagnosed by both transthoracic and transesophageal echography. Recent meta-analysis based on indirect comparisons between LMWHs and DOACs suggest that DOACs have similar efficacy to LMWHs for the treatment of cancer with venous thrombo-embolism [START_REF] Posch | Treatment of venous thromboembolism in patients with cancer: A network meta-analysis comparing efficacy and safety of anticoagulants[END_REF].

However, these meta-analyses have studied the efficacy of DOACs on the prevention of VTE. In this case, the patient had a right lower extremity deep vein thrombosis complicated by pulmonary embolism, but she also presented marantic endocarditis related to the lung cancer. Indeed, marantic endocarditis can occur in cancer patients, related to a multifactorial hypercoagulability state: procoagulant factors synthetized by cancer cells, thrombogenic treatment and the inflammatory status of the host [START_REF] Varki | Trousseau's syndrome: multiple definitions and multiple mechanisms[END_REF]. They are frequently observed in evolved and multi-metastatic cancer, and especially in adenocarcinoma lung cancer. Histologically, they are composed of fibrinous and platelet thrombus without inflammatory reaction [START_REF] Bot | Non-infective endocarditis][END_REF]. In fact, we know that cancer patients have a high risk of developing arterial thrombosis with arterial thromboembolism (ATE), caused by both upregulation of coagulation and activation of platelets [START_REF] Varki | Trousseau's syndrome: multiple definitions and multiple mechanisms[END_REF]. Marantic endocarditis is an example of a rare arterial thrombosis which can lead to arterial embolism, and particularly cerebral embolism with ischemic strokes. In the case of ATE in cancer patients, the efficiency of DOACs is unclear. In fact, DOACs are direct thrombin or factor Xa inhibitors which impact the coagulation cascade, but they are insufficient for preventing platelet thrombus. In this case, the patient presented recurrent ischemic strokes with curatives doses of rivaroxaban (first neurologic episode) then apixaban (second neurologic episode), yet she presented no complications with UFH or LMWHs. A possible explanation for this phenomenon is the insufficiency of DOACs to prevent the formation of platelet thrombus and thus arterial thrombus and ATE, especially in marantic endocarditis. At the opposite, heparin therapies have pleiotropic effects, exerting several antithrombotic effects, beyond their ability to irreversibly inactivate both activated factor Xa and thrombin [START_REF] Varki | Trousseau's syndrome: multiple definitions and multiple mechanisms[END_REF]. Those pleiotropic effects may help prevent ATE events in patients with cancer. Furthermore, randomized controlled trials have only studied the efficacy of edoxaban and rivaroxaban for the treatment of cancer with venous thrombo-embolism.

In cancer patients with marantic endocarditis, the treatment is those of the underlying neoplasia and curative anticoagulation with heparin [START_REF] Bot | Non-infective endocarditis][END_REF]. The role of DOACs in this context still remains to be defined. Two other cases of recurrent cerebral embolism in cancer patients with marantic endocarditis treated with DOACS have been described in the literature [START_REF] Mantovani | A first described case of cancer-associated non-bacterial thrombotic endocarditis in the era of direct oral anticoagulants[END_REF][START_REF] Shoji | Nonbacterial Thrombotic Endocarditis Due to Primary Gallbladder Malignancy with Recurrent Stroke Despite Anticoagulation: Case Report and Literature Review[END_REF]. Our case report enriches the scarce literature on the subject. We conclude that one should systematically consider marantic endocarditis in stroke patients related cancer and eliminate DOAC treatment to prevent ischemic stroke in this context.

Conclusions

Neoplasia associated with VTE and ATE can be responsible for ischemic stroke. The association of VTE and ischemic stroke in cancer patients does not exclude the presence of an ATE phenomenon.

Marantic endocarditis diagnosis must be considered particularly in the context of recurrence of ischemic stroke on direct oral anticoagulant therapy.

Figures legendFigure 1 :

 1 Figures legend

Figure 2 :

 2 Figure 2: PET-scan: hypermetabolic pulmonary nodule associated with hypermetabolic

Figure 3 :

 3 Figure 3: Transesophageal echography: aortic valvular thickening (A) without vegetation and

Acknowledgments

The authors wish to thank Abby Cuttris for critical reading of the manuscript.

Conflict(s) of Interest

The Authors declare that there is no conflict of interest.