Khaled Medini
email: khaled.medini@emse.fr

Thorsten Wuest

Elham Jelodari

David Romero

Valérie Laforest

and agile manufacturing and service operations: Lessons from COVID

Keywords: Customer-Centricity, Sustainability, Mass-Customization, Decision Support System, Portfolio Management, Product Configurator, Operations Management, Production Ramp-Up

This paper reports on a research work conducted within the framework of the SUSTAIN project aiming to unleash and operationalize the potential of customer centricity and sustainability requirements. More specifically, the paper presents a Decision Support System (DSS) intended to support the alignment of aggregate production planning with customer demand during the production ramp-up phase. The DSS consists of a mathematical model and an online product configurator. This will ultimately lead to a revised product portfolio and better align it with the market requirements before moving to series production.

Introduction

Project and operations management have evolved over the last decades in response to environmental and societal challenges and recent technological advancesprior to and aligned with the digital transformation of the manufacturing industry. Customers, whether businesses (B2B) or individual consumers (B2C) are demanding more customization of products, services, and solutions in general. To meet these market requirements, businesses are increasingly faced with the problem of "how diversifying their goods and services offerings to meet a wide range and variety of customer needs in a sustainable way". However, this diversification naturally leads to significantly increased internal complexity linked to the explosion in the volume of data to be processed, but also to the number of different resources and capabilities that must be implemented to ensure a satisfactory diversity of the offer. In addition, there is a need to shorten the development and commercialisation time for customized products and/or services to cope with market volatility and competition that goes beyond the traditional price war [START_REF] Blecker | Complexity and Variety in Mass Customization Systems: Analysis and Recommendations[END_REF][START_REF] Salvador | Cracking the Code of Mass Customization[END_REF][START_REF] Lyons | Customer-Driven Supply Chains From Glass Pipelines to Open Innovation Networks[END_REF].

At the same time, alarming debates on climate change have affected the industrial landscape in the last decades in different ways. International agreements and government policies are driving forces behind the consideration of the environmental impact of industrial activities. Moreover, sustainable systems are characterized by the interactions that affect the economic, social, and environmental sustainability of the enterprisethe so-called: Triple Bottom Line (TBL) [START_REF] Sarkar | A Measure of Product Sustainability Based on Triple Bottom Line[END_REF]. These interactions take place at several levels within and between enterprises and have been the subject of several studies in the scientific and grey literature [START_REF] Labuschagne | Assessing the Sustainability Performances of Industries[END_REF][START_REF] Ageron | Sustainable Supply Management: An Empirical Study[END_REF].

Joint fulfilment of sustainability objectives and customer requirements become a key for the long-term survival of organisations [START_REF] Pardo-Jaramillo | Mapping Research on Customer Centricity and Sustainable Organizations[END_REF]. However, the synergies between these two objectives are not sufficiently exploited and work in this field is rare [START_REF] Hora | Designing Business Models for Sustainable Mass Customization: A Framework Proposal Designing Business Models for Sustainable Mass Customization[END_REF][START_REF] Baldassarre | Bridging Sustainable Business Model Innovation and User-driven Innovation: A Process for Sustainable Value Proposition Design[END_REF]. This paper reports on research work conducted within the framework of the SUSTAIN project aiming to support the alignment of aggregate production planning with customer demand during the production ramp-up phase. The developed DSS consists of a mathematical model and an online product configurator. This will ultimately lead to a revised product portfolio and better align it with the market requirements before moving to series production.

The remainder of the paper is organised as follows, Section 2 reviews the literature highlighting the synergies between customer-centricity and sustainability, Section 3 presents the developed DSS, and Section 4 discusses the proposed DSS and provides research perspectives.

Related Literature

Value Co-Creation

The concept of value has evolved from a perception based on the resource management theory with classical economists [START_REF] Bowman | Value Creation versus Value Capture: Towards a Coherent Definition of Value in Strategy[END_REF], where value is created through a transactional model, to a more complete positioning integrating the customer [START_REF] Prahalad | Co-Creating Unique Value with Customers[END_REF]. Valuein the eyes of a customerrepresents a compromise between perceived benefits and sacrifices associated with a product and/or service offering [START_REF] Prahalad | Co-Creating Unique Value with Customers[END_REF]. This trade-off depends on value attributes like quality, level of customization, and price. In this approach, which emphasises a subjective perception of value for the customer, the Service-dominant logic (SDL) emphasises the relational dimension of value, for instance, the value-in-use that results from the consumption of a service by the customer [START_REF] Vargo | Service-Dominant Logic: Continuing the Evolution[END_REF].

Over the last two decades [START_REF] Bocken | Value Mapping for Sustainable Business Thinking[END_REF][START_REF] Aquilani | The Challenging Transition to Bio-economies: Towards a New Framework Integrating Corporate Sustainability and Value Co-creation[END_REF], the concept of value has increasingly reflected the dimensions of sustainability. In this sense, creating sustainable value means generating economic, environmental, and social benefits associated with an industrial activity [START_REF] Bocken | Value Mapping for Sustainable Business Thinking[END_REF]. However, there isno consensuson what sustainable value actually is [START_REF] Bocken | Value Mapping for Sustainable Business Thinking[END_REF]. A prevailing perception is based on the principle of the Triple Bottom Line (TBL), which extends the value definition from a business perspective to people, planet in addition to profit. More recently, the evolution towards the notion of value co-creation has made it possible to extend the scope of value, which now involves additional stakeholders beyond only the supplier and the customer (e.g., subcontractors), thus forming a more appropriate, yet complex value network [START_REF] Aquilani | The Challenging Transition to Bio-economies: Towards a New Framework Integrating Corporate Sustainability and Value Co-creation[END_REF].

Sustainable mass-customization

Mass-Customisation customisation is a strategy that aims to meet specific customer needs with an efficiency close to that of mass production [START_REF] Pine | Mass Customizing Products and Services[END_REF]. When industrial companies take this route, it presents both opportunities as well as challenges with respect to sustainability in the environmental, social, and economic sense. For example, while standardization and lean management practicesinherited from mass production contribute to optimizing the use of resources and reducing waste, pure customization risks running counter to these objectives when responding to the diversity of individual customer demands [START_REF] Pourabdollahian | Is Sustainable Mass Customization an Oxymoron?[END_REF][START_REF] Medini | Tailoring Performance Evaluation to Specific Industrial Contexts -Application to Sustainable Mass Customisation Enterprises[END_REF]. The challenge is to meet and align the objectives of mass customization and sustainability together. While there are a few selected studies available in the literature addressing the possible complementarities between mass customisation and sustainability, a shortage has been highlighted in terms of design tools and decision support methods that favour the deployment of sustainable mass customisation [START_REF] Pourabdollahian | Is Sustainable Mass Customization an Oxymoron?[END_REF][START_REF] Medini | Tailoring Performance Evaluation to Specific Industrial Contexts -Application to Sustainable Mass Customisation Enterprises[END_REF][START_REF]S-MC-S -Sustainable Mass Customization -Mass Customization for Sustainability[END_REF]. This gap can be partly explained by an implicit assumption that traditional evaluation frameworks are also valid for customised products and their production. However, customisation leads to a diversity in the range of products or services, which adds complexity to the evaluation and requires more adapted evaluation methods [START_REF] Barrows | Cleaner Firms or Cleaner Products? How Product Mix Shapes Emission Intensity from Manufacturing[END_REF][START_REF] Medini | Integrating Sustainability Considerations into Product Variety and Portfolio Management[END_REF]. The S-MC-S (Sustainable Mass Customizationmass customization for Sustainability) project promoted the concept of sustainable mass customization and resulted in a set of decision support tools for implementing it. For instance, a set of indicators and guidelines were proposed to support the design of sustainable mass customized products [START_REF] Osorio | Design for Sustainable Mass-Customization: Design Guidelines for Sustainable Mass-Customized Products[END_REF]. However, the S-MC-S model does not consider the interactions between the various dimensions and requires a large amount of data to be successfully implementeddata that is not always readily available [START_REF]S-MC-S -Sustainable Mass Customization -Mass Customization for Sustainability[END_REF][START_REF] Medini | D3.1 -Assessment Model[END_REF]. The model proposed by [START_REF] Medini | An Approach for Steering Products and Services Offering Variety Towards Economic and Environmental Sustainability[END_REF] aims to strengthen the connection between economic and environmental issues to manage product variety. They propose a mathematical model coupled with simulation and LCA based tools to enable indicators calculation and variety management.

Most of the research works addressing sustainability and customization or customer centricity focus more on the product design phase. This aligns well with the strong impact of the decisions taken during the design phase on the whole product lifecycle. Subsequent phases such as production ramp-up received much less attention despite their potential in reinforcing agility and customer-centricity.

Ramp-up of sustainable mass-customized products

The development of sustainable customer-centric products depends on strategic, tactical, and operational decisions made during all phases of the product or service lifecycle [START_REF] Pardo-Jaramillo | Mapping Research on Customer Centricity and Sustainable Organizations[END_REF]. Most of the work contributing to this issue focuses in particular on the design phase, given its impact on the whole product lifecycle.

However, at this point, the production ramp-up phase is poorly addressed despite its potential impact on successful product commercialisation. In fact, ramp-up presents an opportunity to enhance the sustainability of industrial systems, for example, through decisions on industrial strategy, portfolio management, production resources, skills, and integration of customer feedback [START_REF] Medini | Integrating Sustainability Considerations into Product Variety and Portfolio Management[END_REF][START_REF] Pont | L'Entreprise Industrielle[END_REF].

Ramp-up refers to the phase of the product lifecycle that identifies and deploys the means and methods to move from the study phase to the active production phase. The decisions made during this phase have a strong impact on the company's activities [START_REF] Pont | L'Entreprise Industrielle[END_REF]. These decisions relate to several aspects such as investments and capacity management [START_REF] Hansen | Modelling Ramp-Up Curves to Reflect Learning: Improving Capacity Planning in Secondary Pharmaceutical Production[END_REF][START_REF] Pierné | Economic Assessment of Ramp-Up Strategies in Multi-Variant Production[END_REF], collaboration and information sharing [START_REF] Fjällström | Information Enabling Production Ramp-Up[END_REF], industrial strategy [START_REF] Szabó | Ramp-Up Process Improvement Practices for Time-to-Market Reduction[END_REF], skills and human resources [START_REF] Glock | Production Planning for a Ramp-Up Process with Learning in Production and Growth in Demand[END_REF], and quality management of the product or service in question [START_REF] Colledani | Production Quality Improvement during Manufacturing Systems Ramp-Up[END_REF]. These decisions are more complex in ramp-up than in series production for various reasons such as the uncertainty about customer demands, limited resources, and challenging process control [START_REF] Medini | Integrating Sustainability Considerations into Product Variety and Portfolio Management[END_REF][START_REF] Hansen | Modelling Ramp-Up Curves to Reflect Learning: Improving Capacity Planning in Secondary Pharmaceutical Production[END_REF][START_REF] Fjällström | Information Enabling Production Ramp-Up[END_REF][START_REF] Colledani | Production Quality Improvement during Manufacturing Systems Ramp-Up[END_REF].

A DSS for sustainable portfolio management during ramp-up

DSS overview

The DSS extends a previously published version in [START_REF] Medini | Integrating Sustainability Considerations into Product Variety and Portfolio Management[END_REF] by detailing the models and tools used for capturing customer data and managing product portfolio accordingly. The framework relies on three basic components meant for (i) supporting the alignment of portfolio and planning decisions with customer requirements; (ii) product/service configuration (operational horizon), data maintenance, and quotation process management (operational/tactical horizons); and (iii) variety and portfolio management (tactical horizon) (see Fig. 1).  Capturing Customer Requirementsis supported by a stepwise configuration process allowing customers to customize the product or service to be ordered. The whole process is by a configurator where all feasible combinations are implemented allowing to offer a high variety to the customers. The ease of the configuration process is a key capability to facilitate choice negation for the customers [START_REF] Salvador | Cracking the Code of Mass Customization[END_REF].  The Quotation Processaims to provide economic and environmental indicators estimates based on the selected configurations and on the data about the product, process, and supply chain. Therefore, configuration data employed by the system needs to be regularly updated (e.g., bill-ofmaterials, processes, suppliers, cost data, materials). Economic and environmental indicators should be measurable and significant for decision-making to both the customer and the company [START_REF] Medini | Integrating Sustainability Considerations into Product Variety and Portfolio Management[END_REF].  Portfolio and Variety Managementaims to collect and process data about customer orders (extracted from the configurator) and the product, process, and supply chain data to determine aggregate plans during the production ramp-up phase. It is also concerned with continuously assessing product or service architecture and standardizing as much as feasible the product and the supporting process. Analytical models are the backbone of this part of the framework [START_REF] Medini | Integrating Sustainability Considerations into Product Variety and Portfolio Management[END_REF].

Mathematical model

Several analytical models are available in the literature to support portfolio and/or variety management [START_REF] Medini | Integrating Sustainability Considerations into Product Variety and Portfolio Management[END_REF][START_REF] Medini | An Approach for Steering Products and Services Offering Variety Towards Economic and Environmental Sustainability[END_REF][START_REF] Cooper | New Product Portfolio Management: Practices and Performance[END_REF]. The proposed framework relies on linear programming to determine an aggregate production plan during the ramp-up phase. The ultimate objective is to align planning decisions with customer requirements despite the lack of data during ramp-up. The model is detailed in the following. The objective function uses weighted normalized values of profit and CO2 emissions indicators. For a given product or service variant, the profit is obtained by calculating the difference between the selling price and unit cost (Eq. 1); CO2 emissions are calculated based on a unit CO2 emission associated with the raw material and with the production process (Eq. 2).

𝑃 𝑖 = 𝑆 𝑖 -∑ 𝑏 𝑘 𝑖 × 𝑐 𝑘 𝑘∈𝐵 𝑖 -∑ 𝑎 𝑝 𝑖 × 𝑐 𝑝 𝑝∈𝐴 𝑖
(1)

𝐶 𝑖 = ∑ 𝑏 𝑗 𝑖 × 𝑓 𝑗 𝑗∈𝑀 𝑖 (2)
The normalization of the indicators is enabled by an improved sigmoid function allowing for a pseudo-linear mapping of the original values (values between 𝑥 and 𝑥 𝑚𝑎𝑥 ) [START_REF] De Marsico | NABS: Novel Approaches for Biometric Systems[END_REF] as shown in Eq. 3, with 𝑎 = 2 + √3 and 𝑏 = 7 -4√3.

The normalized values of 𝑃 𝑖 and 𝐶 𝑖 obtained following Eq. 3 are referred to by 𝑛𝑃 𝑖 and 𝑛𝐶 𝑖 . Weights are then assigned to each of the indicators which reflect the importance from the customer point of view.

The weights of the indicators are derived from the configuration data over a given time horizons. The determination of the weights depends on data availability and may use linear multiple linear regression (short/midterm) or moving average (short term).

The objective function, H is presented in Eq. 4, the aim is to maximize it. Eq. 5, 6, and 7 represent, production capacity, demand and positivity constraints, respectively.

𝐻 = ∑ 𝜔 𝑝 . 𝑛𝑃 𝑖 𝑖∈𝑉 -𝜔 𝑐 . 𝑛𝐶 𝑖 (4) ∑ x i𝑡 𝑖∈𝐹 ≤ 𝑃𝐶 𝑡 , ∀𝑡 ∈ 𝑇 (5)
𝐷 𝑖𝑡 -≤ 𝑥 𝑖𝑡 ≤ 𝐷 𝑖𝑡 + , ∀𝑖 ∈ 𝐹, ∀𝑡 ∈ 𝑇 (6)
𝑥 𝑖𝑡 ≥ 0 , ∀𝑖 ∈ 𝐹, ∀𝑡 ∈ 𝑇 (7)

Configuration and Quotation Process

A product configurator refers to a software-based expert system that supports the user in the creation of product or service specifications [START_REF] Haug | Definition and Evaluation of Product Configurator Development Strategies[END_REF]. The literature identified several challenges for the successful development of configurators, such as resource constraints (e.g., time and effort related to configurator development projects), product complexity, IT infrastructure, and organisational burdens (e.g., acceptance within the organisation) [START_REF] Kristjansdottir | The Main Challenges for Manufacturing Companies in Implementing and Utilizing Configurators[END_REF]. Therefore, the development of a (product) configurator should be driven by the requirements of the business and should allow for smooth integration within the company information system as well as work procedures.

The configurator should support a clear configuration process allowing for capturing customer preferences and a transparent quotation process informing the customer of the economic and sustainability implications of the selected configuration. Fig. 2 illustrates the configuration and quotation process in a typical and basic scenario of product customization and ordering. The modelling of the scenario allows for deriving the technical requirements of the configurators.

Fig. 3 shows an excerpt of the graphical interface of the configurator with an illustrative example of a 3D printed customized product. The user is guided through the definition of the product using a set of customization attributes (e.g., colour, material, dimensions). The configurator is linked to a database where all data about material types, unit costs, and environmental characterization factors are available. This allows for providing the customer with a rough quotation (estimate) including price and CO2 emissions associated with the selected configuration of the product highlighting the impact of certain decisions and features. The example is purposely chosen and represents a simple product that allows for a clear depiction of the potential features of the configurator. Several additional features of the configurator are not discussed in this paper since configurator development is ongoing. This version of the configurator extends the work of [START_REF] Rousseau | Configurators as a means to Leverage Customer-Centric Sustainable Systems -Evidence from the 3D-Printing Domain[END_REF], which specifies the general architecture of the configurator and provides a proof of concept. The current version involves the development of a prototype including a database of available product variants and a module for customer order and configuration process treatability.

Discussion and perspectives

In order to ensure long-term competitiveness, a continuous challenge for manufacturing and service companies is to combine customer-centricity with increased sustainability in the environmental, economic, and social sense [START_REF] Pardo-Jaramillo | Mapping Research on Customer Centricity and Sustainable Organizations[END_REF].

The scientific literature related to project and operations management in the broadest sense highlights the lack of research works related to production ramp-up, one of the key stages of the industrialisation phase. This research gap is amplified by the current economic and social context where the development of innovative solutions has become a recurrent activity to meet demand and survive the competition. The context of the current COVID-19 health crisis illustrates the need for agility and rapid adaptation, in quantity and quality, to market changes [START_REF] Ahmad | Reconfiguring and Ramping-Up Ventilator Production in the Face of COVID-19: Can Robots Help[END_REF][START_REF] Vogel-Heuser | Ramp-Up[END_REF]. In this context and at present, production ramp-up has become a key issue for which all means are deployed not only at the level of manufacturing and service companies, but also at the level of public authorities. However, decisions linked to industrialisation and production ramp-up are subject to constraints of uncertainty and urgency. Such decisions must also take into account the priority needs of the market and the specific demands of customers or groups of customers. They also have a social, societal, environmental, and economic impact that must be anticipated.

The proposed approach suggests combining product configurators with mathematical models to align the aggregate production plan based on customer orders during the ramp-up phase to inform decision makers early of the impacts of their choices and thus allow for adaptations before the associated sunk cost becomes too high. This will ultimately lead to a revised product portfolio and to align it with the market requirements before moving to series production. Therefore, the failure risk of product introduction into the market is mitigated through the quick adaptation of the portfolio to sustainability and customer requirements.

The research concerning customer-centric sustainability seems in its infancy. The current paper provides one step forward to operationalize this concept while focusing on the ramp-up phase of the product life cycle. Therefore, there is still much to be desired in terms of improvements and extension of the proposed DSS. First, the technical implementation needs to be finalized and the whole system needs to be tested. This activity is planned for within the short term. A set of customers with different preferences with regards to environmental sustainability will be selected. The configurator will be used to customize and order a 3D printed product. The mathematical model will be used to align the planned production with the demand based on customer past orders. This research is ongoing, and the test is planned for the short term.

Fig. 1 .

 1 Fig. 1. Framework Design (Adapted from [20])

  Notations: 𝑖 product variant 𝑉 set of available variants in the product portfolio 𝐵 𝑖 bill of material of variant 𝑖 𝐴 𝑖 set of operations involved in the realization of 𝑖 𝑀 𝑖 set of materials used to build 𝑖 𝐹 product family 𝑡 ramp-up period 𝑇 set of ramp-up periods 𝑃𝐶 𝑡 total production capacity during period 𝑡 𝐷 𝑖𝑡 lower demand limit of variant 𝑖 during period 𝑡 𝐷 𝑖𝑡 + upper demand limit of variant 𝑖 during period 𝑡 𝑃 𝑖 unit profit margin generated by variant 𝑖 𝑆 𝑖 unit selling price of variant 𝑖 𝐶 𝑖 unit greenhouse gas emissions allocated to variant 𝑖 𝑏 𝑘 𝑖 quantity of purchased component/material 𝑘 used to build one unit of 𝑖 𝑐 𝑘 unit purchasing cost of 𝑘 𝑎 𝑝 𝑖 duration of operation 𝑝 to realize one unit of 𝑖 𝑐 𝑝 unit cost of operation 𝑝 𝑏 𝑗 𝑖 quantity of material 𝑗 used to build one unit of 𝑖 𝑓 𝑘 characterization factor of greenhouse gas emissions related to material 𝑘 𝑛𝑃 𝑖 unit profit generated by variant 𝑖 𝑛𝐶 𝑖 unit CO2 emissions allocated to variant 𝑖 𝑥 𝑖𝑡 decision variable representing production volume of variant 𝑖 during period 𝑡

*Fig. 2 .

 2 Fig. 2. Sequence diagram of the configuration and quotation process

Acknowledgements

This work is supported by the Thomas Jefferson Fund through SUSTAIN project (https://perma.cc/596H-EVYN). The authors would like to thank Salha Medini for the valuable support with the development of the configurator prototype.


domain: spi chim phys

text:
Richard Brédy
email: richard.bredy@univ-lyon1.fr

Marius Hervé

Alexie Boyer

Jeffery M Brown

Isabelle Compagnon

Franck Lépine

Non-ergodic fragmentation of protonated reserpine using femtosecond laser activation

Keywords: Fragmentation, mass spectrometry, reserpine, femtosecond

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

Introduction

Mass spectrometry techniques are widely used for analytical purpose of gas phase ions in the fields of proteomics [START_REF] Aebersold | Mass spectrometry in proteomics[END_REF], for peptide and protein characterization [START_REF] Brodbelt | Ion activation methods for peptides and proteins[END_REF][START_REF] Jonsson | Mass spectrometry for protein and peptide characterisation[END_REF][START_REF] Wysocki | Mass spectrometry of peptides and proteins[END_REF], DNA sequencing [START_REF] Edwards | Mass-spectrometry DNA sequencing[END_REF][START_REF] Köster | A strategy for rapid and efficient DNA sequencing by mass spectrometry[END_REF][START_REF] Tost | DNA analysis by mass spectrometry-past, present and future[END_REF] and carbohydrates [START_REF] Zaia | Mass spectrometry of oligosaccharides[END_REF][START_REF] Han | Mass spectrometry of glycans[END_REF][START_REF] Kailemia | Oligosaccharide analysis by mass spectrometry: A review of recent developments[END_REF][START_REF] Mirgorodskaya | Cracking the sugar code by mass spectrometry[END_REF][START_REF] Schindler | Anomeric memory of the glycosidic bond upon fragmentation and its consequences for carbohydrate sequencing[END_REF]. Based on molecular fragmentation, the analytical methods establish diagnostic fragmentation patterns to provide identification and structural characterization of biomolecules.

Understanding the underlying excitation and fragmentation processes of these biomolecular ions is therefore of importance to manipulate, in a controlled manner, the fragmentation pathways and unambiguously identify biomolecular fingerprints. Based on this knowledge of molecular properties, new strategies or more sophisticated protocols may be elaborated to broaden the sequence coverage.

Various activation methods [START_REF] Brodbelt | Ion activation methods for peptides and proteins[END_REF] have been developed to diversify the accessible fragmentation patterns. Among them, collision induced dissociation (CID) [START_REF] Wells | Collision-induced dissociation (CID) of peptides and proteins[END_REF] is still one of the most common and easiest implemented methods, often used as a reference. CID relies on collisions with a gas of inert atoms that gradually convert part of the kinetic energy of the molecular ions of interest into internal energy leading, in general, to statistical fragmentation. As a result, the observed fragments originate from the cleavage of the weakest bonds after intramolecular energy redistribution. Similar mechanisms occur also, for instance, in infrared multiple photon dissociation (IRMPD) [START_REF] Polfer | Infrared multiple photon dissociation spectroscopy of trapped ions[END_REF] techniques where the vibrational internal energy increase of the ion is due to multiple photon absorption. The weakest bonds prominence in statistical fragmentation appears then as a limitation in the sequence coverage. As many building blocks may not be observed, the potential gain on the structural information is reduced. Moreover, with molecules of increasing size, dissociation thresholds may not be reached as the internal energy is shared among a larger number of degrees of freedom. To overcome these limitations, complementary activation methods have been developed to target processes that involve non-statistical fragmentation pathways. These methods include for example various electron based techniques such as electron capture dissociation (ECD) or electron transfer dissociation (ETD) [START_REF] Breuker | Nonergodic and conformational control of the electron capture dissociation of protein cations[END_REF][START_REF] Lermyte | Radical solutions: Principles and application of electron-based dissociation in mass spectrometry-based analysis of protein structure[END_REF], as well as excitation based on light interaction processes such as ultraviolet photodissociation (UVPD) [START_REF] Brodbelt | Photodissociation mass spectrometry: new tools for characterization of biological molecules[END_REF], high energy photon/VUV/XUV irradiation [START_REF] Bari | Photodissociation of protonated leucine-enkephalin in the VUV range of 8-40 eV[END_REF][START_REF] Giuliani | Extreme ultraviolet radiation: A means of ion activation for tandem mass spectrometry[END_REF], or intense femtosecond laser interaction (fs-LID) [START_REF] Kalcic | Femtosecond laser-induced ionization/dissociation of protonated peptides[END_REF].

With the advent of those activation methods, the question on the validity of the ergodic assumption for the observed fragmentation pathways has been raised. Non-ergodic processes frequently invoked to explain ECD/ETD of protonated peptide cations has been a matter of debates as various experimental results on peptide radical cations could be rationalized using statistical models or abinitio theory without invoking non-ergodic assumption [START_REF] Lermyte | Radical solutions: Principles and application of electron-based dissociation in mass spectrometry-based analysis of protein structure[END_REF][START_REF] Turecek | Peptide radicals and cation radicals in the gas phase[END_REF][START_REF] Laskin | Is dissociation of peptide radical cations an ergodic process?[END_REF]. Both statistical and non statistical fragmentation have been reported in UV photodissociation [START_REF] Grégoire | Statistical vs. non-statistical deactivation pathways in the UV photo-fragmentation of protonated tryptophan-leucinedipeptide[END_REF][START_REF] Byskov | On the photostability of peptides after selective photoexcitation of the backbone: prompt versus slow dissociation[END_REF][START_REF] Julian | The mechanism behind top-down UVPD experiments: Making sense of apparent contradictions[END_REF] and the concept of nonergodic molecules was probed in femtosecond laser activation experiment [START_REF] Diau | Femtosecond activation of reactions and the concept of nonergodic molecules[END_REF][START_REF] Sølling | The non-ergodic nature of internal conversion[END_REF].

In the case of intense femtosecond laser interaction, a considerable amount of energy can be deposited in a molecule in a very short time. This especially leads to molecular ionization through the simultaneous absorption of more than one photons or tunnelling. The created radical may then fragment in a non-ergodic way, i.e., the timescale for the ultrafast bond cleavage does not allow for complete energy redistribution over the internal degrees of freedom of the ion. For instance, evidences for non-ergodic processes in mass-spectrometry experiments were observed using short laser pulse activation [START_REF] Kalcic | Femtosecond laser-induced ionization/dissociation of protonated peptides[END_REF][START_REF] Diau | Femtosecond activation of reactions and the concept of nonergodic molecules[END_REF][START_REF] Sølling | The non-ergodic nature of internal conversion[END_REF][START_REF] Kalcic | Mechanism elucidation for nonstochastic femtosecond laser-induced ionization/dissociation: From amino acids to peptides[END_REF][START_REF] Neidel | Femtosecond laserinduced dissociation (fs-LID) as an activation method in mass spectrometry[END_REF][START_REF] Reitsma | Femtosecond laser induced ionization and dissociation of gas-phase protonated leucine enkephalin[END_REF]. The ultrashort activation time appears as a key parameter. It is suggested that the ultrafast creation of the radical cations plays a role and can induce selective reactions on a time scale faster that intramolecular energy redistribution.

Experiments involving photoactivation such as in fs-LID require an efficient coupling of the femtosecond laser with the molecular ions source and the mass spectrometer. Electrospray ionization (ESI) sources represent the technique of predilection for producing and transferring a large variety of molecular ions in the gas phase. In the current instruments, which are mostly designed for analytical purpose, ion trapping devices are usually required to compensate for the low ion density, to provide long activation time and to perform MS n experiments. It is therefore not surprising that so far most photo-dissociation experiments have been conducted using ion trapping devices coupled to laser systems with high repetition rate, in the kHz range. The long irradiation time, typically over tens of milliseconds, implies that the trapped molecular ions cloud experiences interaction with more than one laser pulse. Thus, unless special care is undertaken, ions and subsequent fragments may be activated by several laser pulses. By using relatively tight laser focus, the contribution of multiple interactions can be held on a relatively low level and determined statistically, thanks to the mixing of the trap content. However, multiple interaction processes cannot be completely ruled out and difficult to exclude at very high repetition rate. This raises questions when it comes to elucidate the primary fragmentation mechanisms using for example pump-probe schemes. Therefore, investigating the intrinsic processes at play during the very first steps of the fragmentation necessitates to ensure that each ion interacts, at most, with a single laser pulse. To achieve this condition, a cross beam configuration between a beam of molecular ions and a laser beam, without trapping device, may be used. With ion velocity in the order of few tens of m/s, typical interaction region of about 100 µm and laser repetition rate in the kHz range this "on-the-fly" type of experiment ensures that ions interact only once with the laser field, disentangling this interaction from "multiple-pulses" interactions. It is noteworthy that the strength of on-the-fly configuration also offers the possibility to implement techniques that are hard to access when using a trap, such as pump-probe experiments to study, for instance, fragmentation dynamics, measurements in coincidence or of quantities such as electron emission or kinetic energy release. Nonetheless, such on-the-fly spectroscopy is still challenging with molecular ions as it involves the production of a selected m/z parent ions beam with sufficient density coupled to a sensitive detection system over a wide dynamical range even at the signal-to-noise limit. We have performed on-the-fly fs-laser interaction experiment by coupling a femtosecond laser with a triple quadrupole instrument, i.e., without trapping device, to explore fragmentation processes on protonated reserpine (scheme 1). Reserpine is a model molecule, extensively used as a chemical standard to calibrate and evaluate performances of mass spectrometers. Extracted from the root of Rauwolfia species, reserpine is a monoterpene indole alkaloid that has been used as drug treatment for hypertension with potential side effects such as depression [START_REF] Hedgecock | Molecular mechanisms and applications of a reserpine-induced rodent model[END_REF][START_REF] Shamon | Blood pressure-lowering efficacy of reserpine for primary hypertension[END_REF]. With a pentacyclic core structure, the total synthesis of reserpine remains challenging [START_REF] Chen | Reserpine: A challenge for total synthesis of natural products[END_REF]. Yet, reserpine has been used in mass spectrometry mostly from an analytical point of view to assess the fragmentation patterns for fingerprints identification or diagnostics [START_REF] Anderson | Quantitative ionspray liquid chromatographic/tandem mass spectrometric determination of reserpine in equine plasma[END_REF][START_REF] Fournier | Integration of cryogenic ion vibrational predissociation spectroscopy with a mass spectrometric interface to an electrochemical cell[END_REF][START_REF] Iqbal | Simultaneous determination of reserpine, rescinnamine, and yohimbine in human plasma by ultraperformance liquid chromatography tandem mass spectrometry[END_REF][START_REF] Kumar | Identification, characterization and distribution of monoterpene indole alkaloids in Rauwolfia species by Orbitrap Velos Pro mass spectrometer[END_REF][START_REF] Kumar | Structural characterization of monoterpene indole alkaloids in ethanolic extracts of Rauwolfia species by liquid chromatography with quadrupole time-of-flight mass spectrometry[END_REF]. Surprisingly very little is known about the physical properties of this molecule related to, for example, its electronic structure or its relaxation pathways dynamics.

In this work we propose to focus on the first steps of the interaction of a molecule with a single femtosecond laser pulse. We show that on-the-fly fs-laser interaction between protonated reserpine and 800 nm intense laser pulses leads to fragmentation processes triggered either by multiphoton ionization (MPI) or tunnel ionization (TI) depending on the considered laser intensities. The resulting fragmentation pathways are significantly different from the statistical ones observed in CID or by single photon resonant electronic excitation at 266 nm. Identification of some specific fragments sheds new light on the non-ergodic fragmentation occurring following fs-laser activation.

Material and methods

The experimental setup was based on a Xevo TQ-S micro triple-quadrupole instrument (Waters), modified to enable laser injection after the first quadrupole (Q1) [START_REF] Hervé | On-the-fly femtosecond action spectroscopy of charged cyanine dyes: Electronic structure versus geometry[END_REF] (SI Fig. S1). Reserpine (C33H40N2O9, m = 608 a.m.u.) purchased from Sigma-Aldrich was dissolved in MeOH:H2O 1:1, with 0.1 % of acetic acid, at a concentration of 60 µM. The solution was injected into vacuum by ESI, generating molecular ions in the gas phase. The ions of interests, here protonated reserpine, were selected according to their mass-to-charge ratio at m/z 609 using Q1 (MS1) and mass spectra were measured by scanning the last quadruple (Q2, MS2). Collision induced experiments were performed with argon gas in the T-wave collision cell (CC) at P = 4 × 10 -3 mbar, and collision energy voltage from 0 to 30 V.

For fs-laser experiments, ion activation was performed using a femtosecond laser delivering 800 nm (hν = 1.55 eV), 25 fs pulses, with an energy of 2 mJ at 5 kHz. The laser beam was injected orthogonally to the molecular ion beam and focused at the exit of the first quadrupole using a f = 1 m focal lens resulting in a calculated beam diameter of 130 µm in the interaction region. At 800 nm mass spectra were recorded for laser intensities between 0 and 3.8 × 10 14 W/cm 2 (maximum energy per pulse = 640 µJ). Additionally, pulses at 400 nm (hν = 3.1 eV) and 267 nm (hν = 4.65 eV) were produced by secondand third-harmonic generation (SHG and THG) from the 800 nm primary beam. Due to low conversion efficiency of the non-linear crystals used for SHG (about 15 %) and THG (about 2 %) mass spectra were recorded, after appropriate filtering, for a single energy per pulse at 400 nm (30 µJ/pulse, 1.5 × 10 13 W/cm 2 , 60 fs pulse duration) and at 267 nm (5 µJ/pulse, 2.8 × 10 12 W/cm 2 , 80 fs pulse duration). The calculated beam diameter in the interaction region at 400 nm and 267 nm was 90 µm and 75 µm, respectively. Typical ion velocity was on the order of 100 m/s in the interaction region delimited by the laser beam diameter at the focal point. Therefore, the ions were traveling several millimetres between two laser pulses separated in time by 200 µs. This insures that an ion was interacting only once with the laser field. The overall background pressure inside the instrument was about 10 -5 mbar, except inside the collision cell, where an argon pressure of 4 × 10 -3 mbar is necessary to guide the ions with a good transmission efficiency toward MS2, and to perform CID. For fs-laser experiments, the collision energy voltage was kept to 0 V to avoid interplay between CID and fs-laser interaction signals.

Results and discussion

Typical CID mass spectra with collision energy voltage set to 25 V and to 0 V are presented in Fig. 1.

They are compared to mass spectra obtained using fs-laser interaction at 267 nm (5 µJ/pulse, 2.8 × W/cm 2 ), 400 nm (30 µJ/pulse, 1.5 × 10 13 W/cm 2 ) and 800 nm (640 µJ/pulse, 3.8 × W/cm 2 ) (see also SI Fig. S3 to S6). Each spectrum is normalized to the total intensity.

Fragmentation yields (Ifragment/Itotal) for major or specific fragments are presented in supplementary material (SI Table ST1). Fragments observed in CID mass spectra as a function of collision energy (SI Fig. S7 to S9) correspond to those observed in the literature [START_REF] Kumar | Identification, characterization and distribution of monoterpene indole alkaloids in Rauwolfia species by Orbitrap Velos Pro mass spectrometer[END_REF][START_REF] Kumar | Structural characterization of monoterpene indole alkaloids in ethanolic extracts of Rauwolfia species by liquid chromatography with quadrupole time-of-flight mass spectrometry[END_REF]. Below 30 V collision energy voltage, the dominant fragments are observed at m/z 174, 195, 397 and 448 (Fig. 1a). In particular, the fragment at m/z 397 corresponds to the bond cleavage after the carboxyl moiety (dash black bar in scheme 1), following nuclear rearrangements to give closed-shell products. The fragment at m/z 195, which corresponds to the trimethoxybenzoyl group (TMB), results from the fragmentation after the carbonyl moiety of the ester bridge (dash red bar in scheme 1). MS n spectra of protonated reserpine in trap have shown that fragments at m/z 174 and 448 are generated due to C-ring cleavage via retro Diels Alder mechanism [START_REF] Kumar | Identification, characterization and distribution of monoterpene indole alkaloids in Rauwolfia species by Orbitrap Velos Pro mass spectrometer[END_REF]. Breakdown curves extracted from CID mass spectra are presented in Fig. 2, showing the branching ratio dependence of the different fragmentation pathways with the collision energy. As collision energy voltage increases, smaller fragments appear due to higher excitation energy distribution and possible re-excitation of precursor ions as shown by the bell shape of the breakdown curves. Comparison of fs-laser interaction mass spectra at 267 nm, 400 nm and 800 nm show drastic changes in the fragmentation pattern as a function of the laser wavelength (Fig. 1c-e). It should be recalled that due to conversion efficiency in the generation of the 400 nm and 267 nm radiations from the 800 nm primary beam, intensity cannot be keep constant while varying the wavelength. This results in intensity variation of up to two orders of magnitude between 267 nm and 800 nm. Electronic absorption spectra of reserpine in solution exhibit strong absorption between 260 and 380 nm, depending on the solvent [START_REF] Eldaroti | Spectroscopic investigations of the charge-transfer interaction between the drug reserpine and different acceptors: Towards understanding of drug-receptor mechanism[END_REF][START_REF] Sakal | Ultraviolet spectrophotometric determination of reserpine[END_REF][START_REF] Ghosh | Determination of individual proton affinities of reserpine from its UV-vis and charge-transfer spectra[END_REF]. Precisely, two absorption bands have been identified, one at λmax ≈ 295 nm (S1 state) and one at λmax ≈ 270 nm (S2 state) in methanol as well as in 5 N acetic acid [START_REF] Eldaroti | Spectroscopic investigations of the charge-transfer interaction between the drug reserpine and different acceptors: Towards understanding of drug-receptor mechanism[END_REF][START_REF] Sakal | Ultraviolet spectrophotometric determination of reserpine[END_REF], while in aqueous solution the absorption peaks at λmax ≈ 292 and 326 nm [START_REF] Ghosh | Determination of individual proton affinities of reserpine from its UV-vis and charge-transfer spectra[END_REF]. Though absorption bands can shift significantly for liquid or gas phase molecules, it is nonetheless expected that single photon absorption probability of gas phase protonated reserpine is higher at 267nm than at 400 nm, and negligible at 800 nm. The fs-laser interaction mass spectrum at 267 nm presents clear fragmentation pattern with main fragments observed at m/z 397 and 448 (Fig. 1c). Overall, the fragmentation channels at 267 nm are similar to those observed in CID mass spectra, although the branching ratios are different. This suggests that fragmentation at this wavelength is mostly induced by statistical processes, due to resonant electronic excitation followed by internal conversion. Oppositely, absorption probability around 400 nm is weak (Fig. 1d) and no significant fragmentation induced by fs-laser interaction could be observed at this wavelength and peak laser intensity (1.5 × 10 13 W/cm 2 ). The negligible fragment signal observed in the corresponding mass spectrum is attributed mostly to residual fragmentation induced by collision with the background gas as suggested by mass spectra recorded without laser injection and collision energy voltage set to 0 V (Fig. 1b). This supports the absence of a resonant state at 400 nm. Mass spectra at 800 nm unambiguously reveals ion signal induced by laser interaction (Fig. 1e). Due to much higher laser peak intensity (3.8 × 10 14 W/cm 2 ), this signal is attributed to processes involving a large number of photons. It is therefore noteworthy that while some fragments, as m/z 195, are observed as in CID or 267 nm excitation, the 800 nm fs-laser interaction mass spectra present a different pattern with very specific fragments (SI Fig. S3). First of all, the radical dication (m/z 304.5) obtained by photoionization of [Res.H] + is observed. This indicates that ionization of protonated reserpine can occur, leaving the molecule intact on the observation time window (on the order of few tens of milliseconds). It is noticeable that peak at m/z 304.5 is not observed in CID mass spectra nor at 267 nm. Secondly, the main fragment at m/z 414 observed in the 800 nm mass spectrum is observed with much lower probability in CID, which instead exhibits fragment at m/z 413 (see insets in Fig. 1 and SI Fig. S3). The fragment at m/z 414 corresponds exactly to the complement moiety of the TMB fragment (m/z 195), following cleavage of the O-R bond in the ester bridge (red bar in Scheme 1). As the complementary fragments at m/z 195 and 414 share the same bond cleavage, this strongly suggests that ionization of protonated reserpine can be followed by specific fragmentation of the radical dication [Res.H] •2+ (Fig. 3a). It is suggested that m/z 414 fragment results from Coulomb repulsion of excited reserpine dication formed by ionization. Following the ultrafast ionization of protonated reserpine, the created radical dication might either remain stable with a lifetime on the order of few tens of milliseconds or fragment via both statistical and non-statistical processes depending on the internal energy. The reserpine dication carries two positive charges, one at the protonation site and one hole. In the case of fragmentation, the two charges may be shared between the two fragments at m/z 195 and 414 and fragmentation could occur without nuclear rearrangement, the largest moiety being for instance the protonated fragment (m/z 414) while the complement moiety (m/z 195) bears the hole. These processes are likely to occur in dication molecules due to the strong Coulomb repulsion, and may occur on ultrafast timescales.

The difference in fragmentation pathway of protonated reserpine leading to the different fragments at m/z 413 and 414 is proposed Fig. 3b. As fragment at m/z 414 corresponds to the O-R bond cleavage after the carbonyl group in the ester link, we suggest that dissociation leading to fragment at m/z 413 results from the same bond cleavage but with an additional hydrogen migration to the carbonyl moiety. This process would produce two fragments with closed-shell configurations, which is statistically more favourable and is more likely to occur in CID because of statistical vibrational energy redistribution. Oppositely, fragmentation pathway leading to the fragment at m/z 414 would not imply closed-shell fragments and no nuclear rearrangement is necessary prior to dissociation. Therefore, using 800 nm intense short pulse fs-laser interaction on protonated reserpine can create radical species that result in different fragmentation patterns than statistically-activated protonated reserpine. In intense fs-laser field, the ionization mechanism depends on the laser wavelength and intensity as well as on the ionization potential of the molecule. In strong field ionization processes of atomic gas information on the ionization regime is provided by the value γ of the Keldysh parameter [START_REF] Posthumus | The dynamics of small molecules in intense laser fields[END_REF][START_REF] Keldysh | Ionization in the field of a strong electromagnetic wave[END_REF][START_REF] Amini | Symphony on strong field approximation[END_REF].

Briefly, in the weak field regime (intensity << 10 14 W/cm 2 , γ >> 1), multiphoton ionization as well as above-threshold ionization are the dominant processes. The ionization yield Y increases with the laser intensity and typically follows a power law Y ∝ I n , with the exponent n being related to the number of absorbed photons. In the strong field regime (intensity in the order or above 10 14 W/cm 2 , γ << 1), the ionization is driven by tunneling effect and the ionization yield reaches a saturation value. In order to understand the underlying process involved in this current work, the evolution of the fragmentation yield has been recorded as a function of the laser pulse intensity for 800 nm excitation wavelength.

The result is presented in Fig. 4 for the main fragments, for laser intensities between 3.5 × 10 13 W/cm 2 and 3.8 × 10 14 W/cm 2 . These intensities cover the range from resonant multiphoton ionization to tunnel ionization regime (1.4 > γ > 0.4, for IP = 8 eV). In the double-logarithmic representation of Fig. 4, a similar trend is observed for all the ion yields that increase with the increasing laser peak intensity.

For intensities between 3.5 × 10 13 W/cm 2 and 1.2 × 10 14 W/cm 2 , the increase of the ion yields follows a power law as shown for the dication (m/z 304.5, n = 3.1 ± 0.1), fragments at m/z 195 (n = 3.1 ± 0.8) and 414 (n = 3.2 ± 0.3). Above 1.2 × 10 14 W/cm 2 , the ion yields deviate from the power law toward a saturation value which indicates that focal volume and tunnelling effects start to become nonnegligible [START_REF] Reitsma | Femtosecond laser induced ionization and dissociation of gas-phase protonated leucine enkephalin[END_REF][START_REF] Posthumus | The dynamics of small molecules in intense laser fields[END_REF]. This laser intensity dependence suggests that a transition from MPI to TI processes is indeed observed in the intensity range considered in this work. It is noteworthy that in this laser intensity range, ionization of protonated reserpine occurs and could lead to non-ergodic fragmentation. This is in line with the idea that ultrafast ionization is one of the key parameters that could trigger specific fragmentation.

To our knowledge, the ionization potential of protonated reserpine has neither been measured nor calculated. It is expected to lie above the IP = 7.88 eV of the neutral reserpine [START_REF] Slifkin | Measurement of ionization potentials from contact charge transfer spectra[END_REF] due to Coulombic effect of the added proton and/or structure modification, this was for instance observed in the case of polypeptides [START_REF] Budnik | Ionization energies of multiply protonated polypeptides obtained by tandem ionization in Fourier transform mass spectrometers[END_REF][START_REF] Giuliani | Structure and charge-state dependence of the gas-phase ionization energy of proteins[END_REF]. In 800 nm multiphoton ionization, at least 5 photons would be required to reach this ionization potential (5 × hν = 7.75 eV ~ 7.88 eV). However, the number of photons (3 to 4) extracted from the slope of the reserpine dication m/z 304.5 is lower. This is a characteristic signature of the existence of an intermediate electronic state lying below the IP, such excited electronic states being resonant with a multiple of the fundamental 800 nm photon energy. Consequently, the production of reserpine dication at 800 nm may be influenced by resonance-enhanced multiphoton ionization (REMPI). In that case, the number of photons deduced from the slope of the dication yield in Fig. 4 would reflect the probability to excite the intermediate state from the ground state (S0) followed by a transition to the continuum. This is consistent with the absorption band at λmax ≈ 270 nm, which may be resonant with three photons absorption at 800 nm. This suggests that the intermediate state plays a role in both 267 nm and 800 nm excitation of protonated reserpine, involving one 267 nm photon (hν = 4.65 eV formed by third-harmonic generation) or three 800nm photons (hν = 1.55 eV). At 800 nm, due to higher intensity, this resonant intermediate electronic state would be populated by 3 photon absorption (3 × hν = 4.65 eV), immediately followed by the absorption of at least two or three photons (2 × hν = 3.1 eV) to reach the ionization potential of the protonated reserpine from the intermediate excited state (4.65 + 3.1 = 7.75 eV ~ IP) (see Fig. 5). Therefore, the power law exponents in the low intensity range strongly support the assumption of an intermediate excited electronic state involved in the ionization process. The major fragment at m/z 414 appears to be specific to 800 nm fs-laser activation method, while it is almost negligible with CID method. However, the fragment at m/z 413, which differs by only one a.m.u., is observed with substantial intensity in both mass spectra. It is therefore noticeable that the yield ratio R = Y414/Y413 ~3.3 is nearly independent of laser intensity in the 800 nm experiments while it is inverted and changes drastically with collision energy in CID (see green and purple curves in Fig. 2 and4). From the ion yield evolution of fragments at m/z 195 and 414 in Fig. 4, the same apparent number of photons (n≈3) is required to induce fragmentation as to ionize the protonated reserpine. This suggests that the fragmentation pathway of protonated reserpine that leads to fragment m/z 414 cannot be explained by statistical excitation of the molecule, as it would be the case in CID, and supports the proposed fragmentation scheme of Fig. 3. In laser interaction at 266 nm and in the MPI regime at 800 nm, due to the excitation of the intermediate resonant state, the probability to ionize or to dissociate protonated reserpine is determined by the probability to absorb extra photons from this excited state. While the intermediate state is involved in both 266 nm and 800 nm excitations, the intensity-driven single-versus multiphoton process leads to different fragmentation pathways. At 266 nm, no supplementary absorption from the excited state is observed and internal vibrational energy redistribution from the intermediate state induces statistical fragmentation. At 800 nm, ionization occurs because of additional photon absorption from the intermediate state, and is eventually followed by non-ergodic fragmentation. The implication of the intermediate state in the fragmentation of protonated reserpine mays also explain the absence of fragmentation observed at 400 nm (hν = 3.1 eV) with laser intensity too low to reach the multiphoton regime.

Regarding fragmentation at 800 nm, it is noteworthy that the mass spectrum does not show strong variations within the intensity range of this study. The low ion yield values in Fig. 4 and their evolution as a function of laser intensity support the interpretation that fragments are generated mostly from the dication and not from the monocation.

Conclusions

Mass spectra of protonated reserpine were obtained using on-the-fly fs-laser/MS experiment at 267 nm, 400 nm and 800 nm and compared with CID activation method. In contrast to experiments based on ion trapping devices, the on-the-fly experimental configuration may favour elucidation of the primary molecular processes involved in the fragmentation mechanisms by allowing, for example, dynamics measurements in pump-probe experiments. Mass spectra at 800 nm fs-laser interaction exhibits very specific fragmentation patterns as well as ionization of the protonated reserpine over a wide range of laser intensities. Activation at 267 nm leads to fragments similar to CID while negligible fragmentation induced by 400 nm fs-laser interaction is observed at the considered laser intensity. The specific fragment m/z 414 observed at 800 nm fs-laser activation is interpreted as due to non-ergodic process triggered by the ultrafast ionization of the protonated reserpine. This ionization can be initiated either by multiphoton or tunnel ionization. Due to the ultrafast increase of the charge, the formed radical dication can fragment through different major channels that differ and may occur much more rapidly than those observed by statistical energy redistribution processes as in CID or 267 nm activation. Interpretation of the results involves the excitation of an intermediate resonant state of protonated reserpine for both 267 nm and 800 nm activation. At the considered laser intensity, single photon absorption at 267 nm preferentially leads to internal energy redistribution while at 800 nm the excited state acts as an intermediate state in the multiphoton ionization process. This single vs many photon aspect suggests that different fragmentation patterns may be laser-intensity-driven. These results could contribute to comprehend the mechanisms involved in analytical methods based on short intense laser interaction. The key point is related to the ultrafast ionization of the protonated molecule that unveils new fragmentation channels due to the Coulomb repulsion between the proton site and the extra-created charge. By understanding the underlying mechanisms involved in such activation methods, specific fragmentation channels may be controlled to help unravelling molecular fingerprints for analytical purpose. These results illustrate the use of ultrafast processes to control bond cleavages in the goal of unambiguously elucidate the molecular structures. Beside the analytical interest, the observation of such specific fragmentation also offers the possibility to study the dynamics of the involved excited states. This kind of studies could unequivocally provide conclusion on the non-ergodic nature of the involved processes and may be of help towards a better understanding of reserpine interactions such as oxidation [START_REF] Fournier | Integration of cryogenic ion vibrational predissociation spectroscopy with a mass spectrometric interface to an electrochemical cell[END_REF] or in drug receptor mechanism via charge transfer [START_REF] Eldaroti | Spectroscopic investigations of the charge-transfer interaction between the drug reserpine and different acceptors: Towards understanding of drug-receptor mechanism[END_REF].

Scheme 1 .

 1 Scheme 1. Protonated reserpine [Res.H] + (C33H40N2O9.H, m/z 609). The dash red bar indicates the O-R

Fig. 1 .

 1 Fig. 1.Comparison of CID and fs-laser mass spectra of protonated reserpine (m/z 609). CID with collision energy voltage set to a) 25 V and b) 0 V, fs-laser interaction at c) 267 nm (5 µJ/pulse, 2.8 × 10 12 W/cm 2 ), d) 400 nm (30 µJ/pulse, 1.5 × 10 13 W/cm 2 ) and e) 800 nm (640 µJ/pulse, 3.8 × 10 14 W/cm 2 ). Insets show the fragments at m/z 413 -414. At 800 nm intact doubly charged

Fig. 2 .

 2 Fig. 2. CID breakdown curves as a function of collision energy voltage: a) for the major fragments at

Fig. 3 .

 3 Fig. 3. Proposed fragmentation pathway of protonated reserpine [Res.H] + : a) after 800 nm fs-laser

Fig. 4 .

 4 Fig. 4. Ion yield a function of peak intensity for 800 nm fs-laser pulse. Lines: power law fit Y ∝ I n for

Fig. 5 .

 5 Fig. 5. Schematic interpretation of protonated reserpine [Res.H] + excitation at 800 nm (MPI) and

Acknowledgments: This work was supported by CNRS and ANR-16-CE30-0012 "Circé".