Leyla Barakat

MD Maria Jose Torres

PhD Elizabeth J Phillips

PhD Marco Caminati

MD Yoon-Seok Chang

PhD Davide Caimmi

MD PhD Mario Sanchez-Borges

PhD Lanny Rosenwasser

PhD Alain Didier

MD PhD Frédéric De Blay

PhD Jean-François Fontaine

MD Isabelle Bosse

MD Sebastien Lefevre

MD Cintia Bassani

MD Maria De Filippo

MD Igancio Ansotegui

PhD Mario Morais-Almeida

PhD Motohiro Ebisawa

PhD Bryan Martin

Yu-Hor Thong

PhD Pascal Demoly

MD PhD Luciana Kase Tanno

BIOLOGICAL TREATMENTS IN ALLERGY: PRESCRIBING PATTERNS AND MANAGEMENT OF HYPERSENSITIVITY REACTIONS

2

Clinical implications : Biological agents (BA) are becoming essential treatments in allergy, but are not available worldwide. Allergists are not authorised to prescribe them in all countries. BA are generally safe, but severe hypersensitivity reactions can occur requiring guided allergological workup and management.

Biological therapies (BA) are emerging as potential effective treatment for allergic and hypersensitivity disorders (A/H). Four main classes of BA are now (May 2020) approved by US Food and Drug Administration and European Medicines Agency for A/H: Anti-immunoglobulin E (IgE) (Omalizumab)

(1), Anti-interleukin 5 (IL5) (Mepolizumab, Reslizumab) (2), Anti-IL4/13 (Dupilumab) [START_REF] Mullard | FDA approves dupilumab for severe eczema[END_REF] and Anti-IL5 R (Benralizumab) [START_REF] Markham | Benralizumab: First Global Approval[END_REF]. Hypersensitivity reactions (HSR) due to BA can occur with different severity degrees, which hamper their use. New types of HSR have been reported with lack of standardized and guided allergy work-up.

Given the novelty of these therapeutics and new challenges faced by the allergy community, we proposed an international survey, which sought to evaluate different aspects related to BA used in the management of HSR due to these drugs.

A web-based survey was undertaken to reach out the worldwide allergy community by e-mail and social media. The web-questionnaire, in English and in French, was constructed using GoogleDocs® and contained 18 questions covering demographic data from participants, BA prescription and related expenses, frequency of HSR and how they are managed (Online Repository Text). It was circulated for 5 weeks and had anonymous and volunteer standards. We received the support from the French Allergy Syndicate (FAS) to send it to their members. Data are presented for 348 participants from 59 countries of all continents. The countries were aggregated according to world regions: North America (NA), Latin America (LA), Europe (EU), Africa and Middle East (AFR/ME), Asia Pacific (AP). Most of the respondents were from EU (62.6%), 87% were allergists with long-term professional experience, 61% worked in a public institution (Table 1).

BA were prescribed by 78.4% of respondents, once or less than once per week (54.6%). Right to prescribe BA was restricted to 68% of allergists. Almost all allergists in EU did not have the right to issue first prescription BA (96.5%), remarkably in France (91%). The most commonly prescribed BA worldwide was the anti-IgE (78%), followed by anti-IL5 (43.9%) then anti-IL13R-IL4R (36.7%) and anti-IL5R (26.7%). NA recorded a higher rate of prescription of new BA (Table 1). The trends of prescription may follow the dynamic of the commercial availability of the BA in the market.

Expenses for BA were mostly completely covered by national social security (59.7%), depending of the country jurisdiction. They were covered by the patient in 10% of cases and by private insurance for 9.1% of respondents. Cost of BA remains an issue from the public health perspective, it is estimated at $10,000 to $30,000 per year/patient receiving BA. Biosimilars drugs, or highly similar copies of BA, will help reducing costs, but while EU has at least 40 biosimilars approved in 2018, US only has five commercially available [START_REF] Chen | Why Biologics and Biosimilars Remain So Expensive: Despite Two Wins for Biosimilars, the Supreme Court's Recent Rulings do not Solve Fundamental Barriers to Competition[END_REF].

The most reported HSR were local reactions at the site of the injection (74%) followed by anaphylaxis (6.8%) and delayed exanthemas (5.1%). Severe cutaneous adverse reactions were rarely reported (<1%). Although these reactions can be allergic (immediate or delayed), most are irritative and can be managed with symptomatic treatment and tends to decrease in frequency and severity with continuation of the injections.

Respondents relied on published data to manage HSR (45.4%), manly national (34.1%) and local recommendations (10%). Lack of national or regional formal recommendations have been reported in 13.5% of respondents.

For mild HSR, most continued ("treated through") the BA, treated the reaction symptomatically (54.6%) and rarely performed allergy investigations (20.7%). For moderate to severe reactions, most decided for switching for an alternative BA (40.5%), but 31% stopped the BA and switched to a nonbiological treatment. Allergy work-up was carried out by 28% of respondents. Desensitization was considered in 18.9% of cases (Table 2). Existing literature estimates the risk of developing anaphylaxis due to omalizumab by 0.09% and by 0.3% to Reslizumab, most (77%) during the first 2 hours after the administration. The pathophysiology of anaphylaxis remains unclear and it seems that there is no apparent correlation between the severity of anaphylaxis and skin test reactivity or the presence of IgE antibodies. Different anaphylaxis phenotypes and endotypes have been identified [START_REF] Castells | Diagnosis and management of anaphylaxis in precision medicine[END_REF]. However, the treatment of the acute reaction remains the same recommended to anaphylaxis.

Allergy tests were infrequently performed by the participants, but should be encouraged to define the mechanism and drug causality of the HSR. Desensitization should be recommended to proven IgE reactions but the decision should be taken individually. For other reactions, desensitization or drug challenge can be considered depending on the severity of the reactions, and the need for the BA [START_REF] Khan | Hypersensitivity and immunologic reactions to biologics: opportunities for the allergist[END_REF][START_REF] Picard | Current Knowledge and Management of Hypersensitivity Reactions to Monoclonal Antibodies[END_REF][START_REF] Barbaud | How to manage hypersensitivity reactions to biological agents?[END_REF]. Delayed reactions were the less frequent type of HSR in our survey, mainly represented by serum sickness like-reaction causing local or systemic injury. Serum sickness like-reaction have been reported 1 to 5 days after the infusion of omalizumab, presenting fever, arthralgia/arthritis, jaw pain or tightness, erythematous skin eruption, purpura and conjunctival hyperemia. Although serum sickness reactions are typically self-limited, re-administration of the culprit BA should not be considered. Other types of delayed HSR to BA remain rare and limited to case reports.

Our study presents some limitations. The initial sample size was not assessed due to the methodology of dissemination. Although we had a limited number and regional/geographical heterogeneity of responses, the qualitative analysis was prioritized. We had higher proportion of responses from France due to the collaboration with the French allergists' community.

This first worldwide survey assessing real-life data from the allergy community provided a snapshot of patterns of prescription of BA used in A/H and information regarding the management of HSR to BA. Although BA are useful in the management of A/H, its prescription seems to be heterogeneous from the international perspective. In several countries, the prescription of BA is restricted to certain authorized specialties, such as dermatologists, pediatricians and pneumologists. The prescription rights of BA may be related to the recognition of allergy as a full specialty nationally and the region/country specialty developments. For instance, in France, allergy has been recognized as a full specialty only in 2017 and the rights to prescribe BA may follow this process, but it is still not a reality as demonstrated in our survey. Most of HSR due to BA are mild local reactions, but severe HSR can occur requiring guided allergy workup and management. There is a lack of consensus of how to manage these HSR, which led us to suggest a decision tree flowchart (Figure E1), which should be validated in the near future.

The first and last authors contributed to the construction of the document (designed the study, designed the questionnaire, analysed and interpreted the data, and wrote the manuscript). All the authors critically revised and approved the final version of the manuscript and agree to be accountable for all the aspects of the work.

AKNOWLEDGEMENTS:

The authors thank the French Allergy Syndicate for their help disseminating the survey to their members.

LIST OF TABLES

Table 1. Demographic data of respondents and prescription of biological agents (AME Africa/Middle-East, AP Asia-Pacific, EU Europe, LA Latin America, NA North America).

Table 2. Management of hypersensitivity reactions due to biological agents depending on the severity of the reaction (BA = biological agents, HSR: hypersensitivity reaction)

Table 1 .

 1 Demographic data of respondents and prescription of biological agents (AME Africa/Middle-East, AP Asia-Pacific, EU Europe, LA Latin America, NA North America).

	Characteristics	NA %	LA %	EU %	AME %	AP %	Total %
		(n/total)	(n/total)	(n/total)	(n/total)	(n/total)	(n)
	Number of responses	22	75	218	16	17	348
	N (%)	(6.3)	(21.5)	(62.6)	(4.6)	(4.9)	(100)
	Specialty 1
	Allergy	100%	92%	85.7%	87.5%	76.4%	87.6%
		(22/22)	(69/75)	(187/218)	(14/16)	(13/17)	(305)
	Clinical immunology	54.5% (12/22)	32% (24/75)	13.7% (30/218)	56.2% (9/16)	11.7% (2/17)	22.1% (77)
	Dermatology 0% (0/22)	0% (0/75)	6.8% (15/218)	0% (0/16)	11.7% (2/17)	4.8% (17)
	Internal Medicine	27.2% (6/22)	6.6% (5/75)	5.9% (13/218)	31.2% (5/16)	5.8% (1/17)	8.6% (30)
	General Medicine 0% (0/22)	1.3% (1/75)	8.2% (18/218)	0% (0/16)	0% (0/17)	5.4% (19)
	Paediatrics 9% (2/22)	13.3% (10/75)	11.9% (26/218)	12.5% (2/16)	35.3% (6/17)	13.2% (46)
	Pneumology 0% (0/22)	4% (3/75)	11% (24/218)	12.5% (2/16)	5.8% (1/17)	8.6% (30)
	Gender
	Female 41% (9/22)	38.6% (29/75)	63.7% (139/218)	50% (8/16)	29.4% (5/17)	54.5% (190)
	Male	59%	61.3%	36.2%	50%	70.5%	45.4%
		(13/22)	(46/75)	(79/218)	(8/16)	(12/17)	(158)
	Age
	≤ 40 years	31.8%	17.3%	40.3%	18.7%	41.1%	33.9%
		(7/22)	(13/75)	(88/218)	(3/16)	(7/17)	(118)
	> 40 years	68.1 % (15/22)	82.6% (62/75)	59.6% (130/218)	81.2% (13/16)	58.8% (10/17)	66% (230)
	Place of work 1
	Public hospital	45.4%	40%	71.5%	43.7%	64.7%	61.4%
		(10/22)	(30/75)	(156/218)	(7/16)	(11/17)	(214)
	Private hospital	36.3% (8/22)	38.6% (29/75)	12.3% (27/218)	37.5% (6/16)	5.8% (1/17)	20.4% (71)

Table 2 .

 2 Management of hypersensitivity reactions due to biological agents depending on the severity of the reaction (BA = biological agents, HSR: hypersensitivity reaction)

		Mild to moderate HSR	Severe HSR
		%	%
		(n/total)	(n/total)
		Actions
	Pursue the same BA and	53.7%	3.7%
	treat the reaction
	symptomatically	(187/348)	(13/348)
		16.6%	40.5%
	Switch of the BA
		(58/348)	(141/348)
	Stop the BA and carry on	8.6%	31.3%
	with non-biological
	treatment	(30/348)	(109/348)
	Allergic investigation (in	21.5%	27.5%
	vivo/in vitro tests)	(75/348)	(96/348)
		12.3%	18.9%
	Desensitization
		(43/348)	(66/348)


Soraya Chapel

Florent Rouvière

Sabine Heinisch
email: sabine.heinisch@univ-lyon1.fr

Comparison of existing strategies for keeping symmetrical peaks in on-line Hydrophilic Interaction Liquid Chromatography x Reversed-Phase Liquid Chromatography despite solvent strength mismatch

Keywords: Two-dimensional liquid chromatography, on-line HILIC x RPLC, solvent strength mismatch, tryptic digest, breakthrough phenomenon

In two-dimensional liquid chromatography, the combination of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) is very attractive due to the complementarity of their separation mechanisms. On-line comprehensive HILIC x RPLC is wellknown to give rise to a large retention space coverage when dealing with ionisable compounds.

However, method development in on-line HILIC x RPLC is challenging due to the reversed solvent strength between both dimensions, which can greatly affect the peak shapes in the second RPLC dimension and thus the separation quality and the method sensitivity. In the present contribution, we compared four strategies designed to avoid this problem: (1) flow splitting, which consists in reducing the injection volume in the second dimension ( 2 D), (2) on-line dilution with a make-up flow and (3) on-line dilution with Active Solvent Modulation (ASM), which both consist in reducing the solvent strength of the injected fractions, and (4) Total Breakthrough Strategy which we recently proposed. Unlike the three preceding strategies, this latter one consists in injecting large volumes of strong solvent in 2 D. The performance of each strategy was evaluated for sub-hour separations of a tryptic digest in on-line HILIC x RPLC. In this work, we considered the critical case for which the same column internal diameters (i.e. 2.1 mm here) are used in both dimensions. Peak capacity, peak shapes and peak intensities were considered for this evaluation. The highest peak capacity could be achieved with Total Breakthrough Strategy while the lowest one with on-line dilution using ASM.

Peak intensities were usually higher with on-line dilution approaches (make-up flow and ASM).

Introduction

On-line comprehensive two-dimensional liquid chromatography (LC x LC) is a powerful tool to deal with highly complex samples. The potential of this technique has been highlighted in various application areas, including, among the most recent ones, proteomics [START_REF] Baghdady | Online Comprehensive High pH Reversed Phase × Low pH Reversed Phase Approach for Two-Dimensional Separations of Intact Proteins in Top-Down Proteomics[END_REF], metabolomics [START_REF] Muller | Predictive kinetic optimisation of hydrophilic interaction chromatography × reversed phase liquid chromatography separations: Experimental verification and application to phenolic analysis[END_REF], natural product research [START_REF] Muller | Application of Kinetically Optimised Online HILIC × RP-LC Methods Hyphenated to High Resolution MS for the Analysis of Natural Phenolics[END_REF], polymer analysis [START_REF] Pirok | Analysis of charged acrylic particles by on-line comprehensive two-dimensional liquid chromatography and automated dataprocessing[END_REF], and pharmaceutical analysis [START_REF] Iguiniz | Quantitative aspects in on-line comprehensive two-dimensional liquid chromatography for pharmaceutical applications[END_REF]. The basic principle of two-dimensional liquid chromatography (2D-LC) is the combination of two chromatographic systems, referred to as dimensions, to expand the level of information on a given sample. To be effective, the two dimensions must provide different selectivities. This is usually achieved by selecting two chromatographic modes with different retention mechanisms. In on-line comprehensive 2D-LC (on-line LC x LC), the entire effluent from the first dimension ( 1 D) is fractionated and continuously transferred to the second dimension ( 2 D) through a switching-valve, often referred to as modulator. Compared to conventional one-dimensional liquid chromatography (1D-LC), a drastic improvement of the resolving power is expected. In theory, the peak capacity should be the product of the peak capacities in the two dimensions. In practice, this is true if the 1 Dseparation is preserved and if the available 2D separation space is fully occupied by peaks (i.e. if the two separation dimensions are fully orthogonal). Apart from a few lucky combinations, such as HIC x RPLC [START_REF] Sarrut | Analysis of antibody-drug conjugates by comprehensive on-line twodimensional hydrophobic interaction chromatography x reversed phase liquid chromatography hyphenated to high resolution mass spectrometry. I -Optimization of separation conditions[END_REF], HIC x SEC [START_REF] Ehkirch | An Online Four-Dimensional HIC×SEC-IM×MS Methodology for Proof-of-Concept Characterization of Antibody Drug Conjugates[END_REF] or IEX x RPLC [START_REF] Vanhoenacker | Comprehensive twodimensional liquid chromatography of therapeutic monoclonal antibody digests[END_REF], for which the mobile phase in 1 D becomes a weak injection solvent in 2 D, the quest for a higher degree of orthogonality between the two dimensions often leads to a decreased compatibility between the mobile phase in 1 D and the separation in 2 D.

A typical example of this duality is the combination of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC). In the past decade, HILIC x RPLC has received a substantial increase in interest for the on-line LC x LC separation of polar and ionisable compounds [START_REF] Montero | Profiling of Vitis vinifera L. canes (poly)phenolic compounds using comprehensive two-dimensional liquid chromatography[END_REF][START_REF] Zhang | A multidimensional analytical approach based on time-decoupled online comprehensive two-dimensional liquid chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry for the analysis of ginsenosides from white and red ginsengs[END_REF][START_REF] Cao | Online comprehensive two-dimensional hydrophilic interaction chromatography×reversed-phase liquid chromatography coupled with hybrid linear ion trap Orbitrap mass spectrometry for the analysis of phenolic acids in Salvia miltiorrhiza[END_REF][START_REF] Martín-Ortiz | Separation of di-and trisaccharide mixtures by comprehensive two-dimensional liquid chromatography. Application to prebiotic oligosaccharides[END_REF]. This growing popularity is driven by the complementarity of the two separation mechanisms which gives rise to high coverage of the two-dimensional retention space [START_REF] Muller | Predictive kinetic optimisation of hydrophilic interaction chromatography × reversed phase liquid chromatography separations: Experimental verification and application to phenolic analysis[END_REF][START_REF] Chapel | Pushing the limits of resolving power and analysis time in on-line comprehensive hydrophilic interaction x reversed phase liquid chromatography for the analysis of complex peptide samples[END_REF].

However, due to the opposite eluent strengths of the two mobile phases, on-line HILIC x RPLC is not guaranteed to succeed. The mobile phase in HILIC typically contains a high percentage of acetonitrile (i.e. > 50%) which is a strong eluent in RPLC. The transfer of acetonitrile-rich fractions from 1 D-HILIC usually greatly impacts the peak shapes in 2 D-RPLC, the solute band being not or partly retained in the 2 D column. This may lead to band broadening, peak distortion, and/or analyte breakthrough.

Breakthrough is a phenomenon that occurs when part of the sample migrates through the column without any retention and elute with the solvent peak whereas the other part undergoes normal retention, which results in the presence of an unretained peak in addition to the expected retained peak. The shape of the retained peak depends on (1) the solute retention in the injection solvent, [START_REF] Muller | Predictive kinetic optimisation of hydrophilic interaction chromatography × reversed phase liquid chromatography separations: Experimental verification and application to phenolic analysis[END_REF] the solute retention in the mobile phase, and (3) the injection volume. These injection effects negatively impact the separation in 2 D by decreasing both the peak capacity and the peak intensity.

Solvent strength mismatch is by far the most critical reported issue in on-line HILIC x RPLC [START_REF] Muller | Predictive kinetic optimisation of hydrophilic interaction chromatography × reversed phase liquid chromatography separations: Experimental verification and application to phenolic analysis[END_REF][START_REF] Muller | Application of Kinetically Optimised Online HILIC × RP-LC Methods Hyphenated to High Resolution MS for the Analysis of Natural Phenolics[END_REF][START_REF] Toro-Uribe | Characterization of secondary metabolites from green cocoa beans using focusing-modulated comprehensive two-dimensional liquid chromatography coupled to tandem mass spectrometry[END_REF][START_REF] Montero | Focusing and non-focusing modulation strategies for the improvement of on-line two-dimensional hydrophilic interaction chromatography × reversed phase profiling of complex food samples[END_REF][START_REF] Sommella | Development of an improved online comprehensive hydrophilic interaction chromatography × reversed-phase ultra-highpressure liquid chromatography platform for complex multiclass polyphenolic sample analysis[END_REF]. To overcome its deleterious impact on the separation in 2 D, a few strategies were proposed over the years. They fall into two broad categories: (i) reducing the injection volume or (ii) reducing the eluent strength of the injection solvent by on-line dilution with a weak solvent. Reducing the volume injected in 2 D can be achieved either by selecting appropriate column internal diameters in each dimension [START_REF] Montero | Profiling of Vitis vinifera L. canes (poly)phenolic compounds using comprehensive two-dimensional liquid chromatography[END_REF] (usually at least twice as large in 2 D) or more commonly by splitting the flow coming from 1 D with a tee-piece and appropriate tubing [START_REF] Sarrut | Optimization of conditions in on-line comprehensive two-dimensional reversed phase liquid chromatography. Experimental comparison with one-dimensional reversed phase liquid chromatography for the separation of peptides[END_REF]. Both approaches inevitably result in increased dilution. On-line solvent dilution consists in diluting the 1 D organic-rich fractions with water before injection in 2 D. Three different methods were reported. The most widespread one makes use of a make-up pump between the 1 D-column and the interface valve [START_REF] Venter | Comprehensive Three-Dimensional LC × LC × Ion Mobility Spectrometry Separation Combined with High-Resolution MS for the Analysis of Complex Samples[END_REF]. The flow rate delivered by the pump can be varied depending on the desired dilution. In 2017, a new valve permitting to achieve dilution inside the valve using restriction capillaries was introduced. It was referred to as "Active Solvent Modulation" (ASM) [START_REF] Stoll | Active Solvent Modulation: A Valve-Based Approach To Improve Separation Compatibility in Two-Dimensional Liquid Chromatography[END_REF][START_REF] Stoll | Development of Comprehensive Online Two-Dimensional Liquid Chromatography/Mass Spectrometry Using Hydrophilic Interaction and Reversed-Phase Separations for Rapid and Deep Profiling of Therapeutic Antibodies[END_REF]. More recently, the so-called "At-Column Dilution" (ACD) was proposed. In this case, dilution is carried out between the valve and the 2 Dcolumn by using an additional pump to empty the sample loop while the 2 D-pump dilutes the transferred fractions [START_REF] Chen | Development of an At-Column Dilution Modulator for Flexible and Precise Control of Dilution Factors to Overcome Mobile Phase Incompatibility in Comprehensive Two-Dimensional Liquid Chromatography[END_REF][START_REF] Chen | Application of the new at-column dilution (ACD) modulator for the two-dimensional RP×HILIC analysis of Buddleja davidii[END_REF]. Regardless of the method, very large volumes which are proportional to the desired dilution ratio, are injected in 2 D. Nevertheless, improvements in both peak shape and peak intensity can be expected from the substantial decrease in eluent strength, which promotes band focusing at the 2 D-column inlet. It should be noted that despite the decrease in solvent strength, very large injection volumes can still have a detrimental effect on peak shapes, especially for less retained compounds. This was highlighted in the context of the separation of pharmaceuticals in 1D-LC [START_REF] Stoll | Evaluation of detection sensitivity in comprehensive two-dimensional liquid chromatography separations of an active pharmaceutical ingredient and its degradants[END_REF]. The combination of both on-line dilution and flow splitting was suggested to lessen this problem [START_REF] Cao | Online comprehensive two-dimensional hydrophilic interaction chromatography×reversed-phase liquid chromatography coupled with hybrid linear ion trap Orbitrap mass spectrometry for the analysis of phenolic acids in Salvia miltiorrhiza[END_REF][START_REF] Stoll | Evaluation of detection sensitivity in comprehensive two-dimensional liquid chromatography separations of an active pharmaceutical ingredient and its degradants[END_REF] but this approach was not compared to on-line dilution alone. Similarly, the use of short trap columns instead of conventional empty loops after on-line dilution with a make-up flow was subjected to numerous developments in recent years [START_REF] Pirok | Recent Developments in Two-Dimensional Liquid Chromatography: Fundamental Improvements for Practical Applications[END_REF]. Trap columns are supposed to reduce the injection volume after on-line dilution by trapping the analytes before being sent to the 2 D column. Despite the increased popularity of trap columns in on-line HILIC

x RPLC [START_REF] Zhang | A multidimensional analytical approach based on time-decoupled online comprehensive two-dimensional liquid chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry for the analysis of ginsenosides from white and red ginsengs[END_REF][START_REF] Martín-Ortiz | Separation of di-and trisaccharide mixtures by comprehensive two-dimensional liquid chromatography. Application to prebiotic oligosaccharides[END_REF][START_REF] Gargano | Reducing Dilution and Analysis Time in Online Comprehensive Two-Dimensional Liquid Chromatography by Active Modulation[END_REF][START_REF] Sommella | Comparison of Online Comprehensive HILIC × RP and RP × RP with Trapping Modulation Coupled to Mass Spectrometry for Microalgae Peptidomics[END_REF], their added value in combination with on-line dilution has still to be demonstrated. Furthermore, the use of 2D-methods with trap columns is questionable for routine analysis since it is more difficult to develop and less rugged considering the risk of sample loss and incomplete recovery.

In a previous work [START_REF] Chapel | Pushing the limits of resolving power and analysis time in on-line comprehensive hydrophilic interaction x reversed phase liquid chromatography for the analysis of complex peptide samples[END_REF], we presented an alternative approach for the separation of peptides in online HILIC x RPLC that we called "Total Breakthrough Strategy" (TBS). This approach relies on the injection of relatively large volumes of strong solvent without flow-splitting and on-line dilution.

Unlike the above strategies, for which breakthrough is unwelcome, our approach is supported by the existence of breakthrough. For large enough injection volumes, it was shown that quite symmetrical peaks could be obtained in 2 D-RPLC. It was also proved that the peak retention time exactly matches the one obtained by injecting a small analyte volume in a weak solvent. Furthermore, despite the presence of breakthrough, quantitative analysis on the retained peak was proved to be quite reliable. Such conclusions could be drawn provided that the injected volume was above a specific critical volume which depends on the analyte, its retention, and its injection solvent [START_REF] Chapel | Pushing the limits of resolving power and analysis time in on-line comprehensive hydrophilic interaction x reversed phase liquid chromatography for the analysis of complex peptide samples[END_REF]. In this case, the area of the retained peak varies linearly with the injected amount which is not the case at an earlier stage of breakthrough (i.e. below the critical volume). The validity of this approach in online HILIC x RPLC was demonstrated under a broad range of conditions in the context of peptide analysis.

The aim of the present study was to compare four of the above-cited strategies, designed to overcome solvent strength mismatch in on-line HILIC x RPLC. In this work, we have considered the critical case for which the same column internal diameter (here 2.1 mm) is used in both dimensions. The compared strategies include (i) the Total Breakthrough strategy (TBS) [START_REF] Chapel | Pushing the limits of resolving power and analysis time in on-line comprehensive hydrophilic interaction x reversed phase liquid chromatography for the analysis of complex peptide samples[END_REF], (ii) the flow splitting strategy (FSS), (iii) the make-up flow strategy (MFS), and (iv) the active solvent modulation strategy (ASMS). Their strengths and weaknesses are identified in the context of an optimized sub-hour separation of a tryptic digest in on-line HILIC x RPLC with a view to select the most appropriate one.

Experimental section

Chemicals and reagents

Acetonitrile (ACN, LC-MS grade) was obtained from Sigma-Aldrich (Steinheim, Germany). Water was purified and deionized in-house using an Elga Purelab Classic UV purification system from Veolia water STI (Décines-charpieu, France). Formic acid (FA, LC-MS grade), ammonium acetate, and ammonium bicarbonate (both analytical reagent grade) were obtained from Fischer scientific (Illkirch, France). DL-1,4-dithiothreitol (DTT, 99%) and iodoacetamide (98%) were obtained from Acros Organics (Geel, Belgium). Trypsin, human serum albumin (HSA), bovine serum albumin (BSA), β-casein, myoglobin, lysozyme, and cytochrome C were all obtained from Sigma-Aldrich (Steinheim, Germany).

Sample preparation

A model sample was obtained by tryptic digestion of six proteins (HSA, BSA, β-casein, myoglobin, lysozyme, and cytochrome C) following a protocol described elsewhere [START_REF] Chapel | Pushing the limits of resolving power and analysis time in on-line comprehensive hydrophilic interaction x reversed phase liquid chromatography for the analysis of complex peptide samples[END_REF][START_REF] Sarrut | Optimization of conditions in on-line comprehensive two-dimensional reversed phase liquid chromatography. Experimental comparison with one-dimensional reversed phase liquid chromatography for the separation of peptides[END_REF].

The aqueous digests were 2-fold diluted with ACN and filtered on 0.22 µm PVDF (polyvinylidene fluoride) membranes before injection in 1 D-HILIC.

Instrumentation

2D-LC experiments were carried out on a 1290 series Infinity 2D-LC system from Agilent Technologies (Waldbronn, Germany). The system includes two high-pressure binary pumps, a thermostated autosampler with a flow-through needle injector, two thermostated column compartments with low-dispersion preheaters, and two diode-array UV absorbance detectors (DAD) with 0.6 µL flow-cells. The switching-valve connecting the two dimensions was either a 2-position /4port duo valve (Fig. S1) or a 4-position/10-port Active Solvent Modulation (ASM) valve (Fig. S2), both configured in backflush mode to minimize extra-column dispersion. In this paper, the first one will be referred to as a standard valve, the second one as the ASM-valve. Depending on the transferred fraction volume, the valves were equipped with two identical sample loops of either 20 µL (0.2 mm ID), 40 µL (0.25 mm ID for the standard valve, and 0.35 mm ID for the ASM valve) or 180 µL (0.35 mm ID). For on-line dilution, the sample loops were connected to the valve through two parking deck (valves used for multiple heart-cutting 2D-LC) using two transfer capillaries of 1.9 µL (0.35 mm ID). A schematic representation of this configuration is shown in Fig. S3. For the make-up flow approach, this setup was used to circumvent the incompatibility of the commercialized 180 µL loops fitting with the standard valve ports. In the case of ASM, not only the comprehensive 2D-LC sample loops are not compatible with the ports of the ASM valve, but this technique also requires backpressure from capillaries between the ASM valve and the multiple heart-cutting valves to function. A pressure release kit was installed between the 1 D outlet and the modulation valve inlet to minimize baseline disturbances coming from 2D-LC valve switching. The dwell volumes and extra-column volumes were respectively 170 µL and 22 µL in 1 D, and 80 µL and 8.5 µL in 2 D (loop volume excluded). For the make-up flow strategy, an additional high-pressure binary pump from an Acquity UPLC I-Class liquid chromatography system from Waters (Milford, MA, USA) was used. The schematic representations of the four different setups used in this work are given in Fig. 1.

The 2D-LC system was hyphenated to an Agilent Q-TOF mass spectrometer (model G6545B) equipped with a Jet Stream electrospray ionization (ESI) source. The 2D-LC-UV system and the additional high-pressure pump were controlled using Agilent OpenLab software and Waters MassLynx software, respectively. The mass spectrometer was controlled using Agilent MassHunter software. The HRMS data were processed using Agilent MassHunter qualitative analysis software.

Chromatographic and detection conditions

For a fair comparison, similar conditions were used for the four presented strategies. The conditions were optimized in a previous study dealing with the on-line HILIC x RPLC separation of peptides in 30 min [START_REF] Chapel | Pushing the limits of resolving power and analysis time in on-line comprehensive hydrophilic interaction x reversed phase liquid chromatography for the analysis of complex peptide samples[END_REF]. The 1 D-HILIC separation was performed using an Acquity BEH HILIC column (50 x 2.1mm; 1.7 µm particles) from Waters (Milford, MA, USA). The column temperature was set at 30°C and the flow rate was 0.05 mL/min. The injected volume was 6 µL. A gradient elution with ACN as solvent A and 10 mM ammonium acetate in water as solvent B (pH of 6.8 in the aqueous phase) was carried out as follows: 0 min (2% B), 30 min (52% B), 32.4 min (2% B), and 50 min (2% B). UV chromatograms were recorded at 210 nm with an acquisition rate of 20 Hz. The sampling time was 0.39 min.

Depending on the studied strategy, the transferred fractions were modified or not before injection in 2 D as discussed in the next section.

The 2 D-RPLC separation was performed using an Acquity CSH C18 column (30 x 2.1 mm; 1.7 µm particles) from Waters (Milford, MA, USA). The column temperature was set at 80°C and the flow rate was 2 mL/min. Water was used as solvent A and ACN as solvent B, both with 0.1% formic acid (pH = 2.7). Initial and final compositions were 1% B and 45% B. The gradient times and the interface conditions were dependent on the studied strategies as discussed in the next section. Their values are summarized in Table 1.

The effluent from 2 D was split using a zero dead volume tee-piece (split 1:2; MS:UV). UV chromatograms were recorded at 210 nm with an acquisition rate of 80 Hz in 2 D. QTOF-HRMS data were acquired in positive ion mode from 100 to 3200 m/z with an acquisition rate of 20 spectra/s. The drying gas temperature and flow rate were 300°C and 11 L/min, respectively. The nebulizer gas pressure was 40 psi. The sheath gas temperature and flow rate were 350 °C and 11 L/min, respectively. The capillary, the nozzle, the fragmentor, the skimmer, and the Oct 1 RV voltages were 3500, 300, 150, 20, and 750 V, respectively.

Calculations

The effective peak capacities, n2D,eff, were calculated from the following relationship:

, = × × 1 + ∆ × 1 + ∆ (1)
∆ is the whole range of elution times, , the average peak width at 4σ (i.e. 13.4% of peak height). α corrects for undersampling in 1 D [START_REF] Davis | Effect of First-Dimension Undersampling on Effective Peak Capacity in Comprehensive Two-Dimensional Separations[END_REF] and γ corrects for partial retention space coverage [START_REF] D'attoma | On-line comprehensive two-dimensional separations of charged compounds using reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography. Part I: Orthogonality and practical peak capacity considerations[END_REF]. In all present 2D-separations, their values were found to be 0.67 for α and 1 for γ (100% coverage), respectively.

The composition of acetonitrile at elution was calculated according to

= + (" #$%&' (" $%$)$&' ) + ( , --. ) in RPLC Or = 100 -0 + (" #$%&' (" $%$)$&' ) + ( , --. )1 in HILIC (2)
Where Cinitial and Cfinal are the initial and final compositions of acetonitrile in the mobile phase, tr is the retention time, tD is the total dwell time, and t0 is the column dead time.

2D-data were processed with Matlab (V7.12.0635).

The following asymmetry factor, Af was considered to assess the peak asymmetry:

2 = 3 4 (3)
With a and b the left and the right half peak width respectively, both measured at 5% of the peak height.

Results and discussion

The objective of this study was to compare four strategies that are currently proposed to reduce broad and distorted peaks and hence to improve the separation in the second dimension of on-line HILIC x RPLC. We considered the peak shape, the peak width, and the peak capacity as the main quality descriptors. The peak intensity and hence the extent to which the analytes were diluted was also compared between the four strategies. Broad and distorted peaks mainly arise from a difference in solvent strength between the organic-rich fraction coming from HILIC and the water-rich mobile phase in RPLC. The four studied strategies included: (i) the Total Breakthrough strategy (TBS), which consists in injecting the entire fractions coming from 1 D so that a situation of Total Breakthrough occurs in 2 D for all peaks [START_REF] Chapel | Pushing the limits of resolving power and analysis time in on-line comprehensive hydrophilic interaction x reversed phase liquid chromatography for the analysis of complex peptide samples[END_REF], (ii) The flow splitting strategy (FSS), which consists in splitting the flow prior to the valve and hence in injecting small volumes of undiluted fractions, and

(iii) the make-up flow strategy (MFS) and (iv) the active solvent modulation strategy (ASMS). Both latter strategies result in injecting large volumes of fractions previously diluted with a weak solvent (usually water). This is carried out with a make-up pump in the first case and the ASM-valve (marketed by Agilent) in the second case. It should be noted that dilution after the switching valve was also recently reported under the name "at-column dilution" [START_REF] Chen | Development of an At-Column Dilution Modulator for Flexible and Precise Control of Dilution Factors to Overcome Mobile Phase Incompatibility in Comprehensive Two-Dimensional Liquid Chromatography[END_REF][START_REF] Chen | Application of the new at-column dilution (ACD) modulator for the two-dimensional RP×HILIC analysis of Buddleja davidii[END_REF]. In this study, we considered the critical case in which the same column internal diameters (i.e. here 2.1 mm) are used in both dimensions. However, it should be noted that FSS with a split ratio of 1/10 should give similar results as those obtained with a ratio of 3 between the internal diameters (e.g. 1 mm in 1 D and 3 mm in 2 D). The studied sample was a tryptic digest of six proteins. Some conditions were specific to the studied strategy, including the gradient time in 2 D and other specific interface conditions. The other conditions, optimized in a previous study [START_REF] Chapel | Pushing the limits of resolving power and analysis time in on-line comprehensive hydrophilic interaction x reversed phase liquid chromatography for the analysis of complex peptide samples[END_REF], are given in the experimental section.

Preliminary considerations and operating conditions for the four strategies

In on-line LC x LC, the continuous transfer of fractions from 1 D to 2 D is achieved by a switching-valve equipped with two sample loops. The interfaces for the four studied strategies are schematically represented in Fig. 1. While a fraction is stored in the first loop, the fraction previously stored in the second loop is injected and analysed in 2 D. The sampling time (i.e. the storage time) represents the total analysis time in 2 D ( 2 t). To essentially maintain the resolution obtained in 1 D while ensuring a sufficient peak capacity in 2 D, the ratio of the sampling time to the 1 D-peak standard deviation ( 1 σ) must be at least 2 [START_REF] Murphy | Effect of Sampling Rate on Resolution in Comprehensive Two-Dimensional Liquid Chromatography[END_REF][START_REF] Horie | Calculating Optimal Modulation Periods to Maximize the Peak Capacity in Two-Dimensional HPLC[END_REF] and sometimes up to 6 in case of sub-hour separations [START_REF] Sarrut | Theoretical and experimental comparison of one dimensional versus on-line comprehensive two dimensional liquid chromatography for optimized sub-hour separations of complex peptide samples[END_REF]. The sampling time and hence the analysis time in 2 D must be short (typically 2 t < 0.5 min for sub-hour separations).

It includes different times. Their repartition is schematically represented in Fig. 2 for the four different strategies.

With TBS, FSS, and MFS, the available gradient time in 2 D is related to these different times according to: and two 2 t0, respectively [START_REF] Grivel | Selection of suitable operating conditions to minimize the gradient equilibration time in the separation of drugs by Ultra-High-Pressure Liquid Chromatography with volatile (mass spectrometry-compatible) buffers[END_REF].

Whereas 2 tD,instrum, 2 tpost and 2 teq cannot be further modified once properly selected, 7 will depend on the loop volume and hence on the selected strategy. Thus, according to Eq.4, the maximum gradient time that can be used will also depend on the selected strategy.

With FSS (Fig. 1b), the flow-rate coming from 1 D ( 1 F), is split with a desired split ratio (zsplit < 1) using a tee-piece placed between the 1 D-column outlet and the valve inlet.

With MFS (Fig. 1c), the dilution of the fractions is achieved by continuously mixing the mobile phase coming from 1 D with a weak solvent delivered by an additional pump. Its flow rate is given by:

= 7 97 = (A B -1) × C = (5)
Where C = is the flow-rate in 1 D and A B is the desired dilution ratio (> 1).

With ASMS (Fig. 1d), dilution of 1 D fractions is achieved with a specific four-position switching-valve equipped with restriction capillaries [START_REF] Stoll | Active Solvent Modulation: A Valve-Based Approach To Improve Separation Compatibility in Two-Dimensional Liquid Chromatography[END_REF]. Different capillaries are available for adjusting the split ratio and thus the dilution. The interface and the way it operates are schematically represented in Figs. S2 and S3 of Supplementary Information. Compared to the standard valve (Fig. S1), the ASM valve displays two additional positions (A and C) and two additional ports (5' and 6'), all involved in the dilution process. The basic principle of ASMS is to make use of the initial 2 D-mobile phase for diluting the fraction volume. It is achieved by using a bypass capillary between the ports 5' and 6' (Figs. S2 andS3b). In position A, the flow coming from the 2 D pump is divided into two distinct paths.

In one path (port 5 to 7), the mobile phase goes through the loop to empty its content, whereas in the other path (port 5 to 4), it goes through the bypass capillary. Depending on the selected bypass capillary, the content of the sample loop is more or less diluted by the 2 D mobile phase when the two paths meet in port 6. After the dilution step, the ASM valve is switched back to the regular flow path (position B) for analysis. The desired dilution factor (A B ), is determined by the capillary dimensions.

Regardless of the strategy, the injection volume in 2 D ( 2 Vinjection) and the composition of the injection solvent ( D E ) can be expressed as:

< D E = C = × × A B × C A 87 (6)
And

D E = " F 3 " G$'H)$I% × (J G$'H)$I% ( C) J G$'H)$I% (7)
With Cdilution, the composition of strong solvent in the diluting solvent.

A B > 1 with MFS or ASMS; =1 otherwise A 87 < 1 with FSS; =1 otherwise It was often shown that the peak injection compression factor, CF, which expresses how much the injection plug is reduced (CF > 1) or broadened (CF < 1) into the column, is directly related to the ratio of the retention factor in the injection solvent (ks) to the retention factor in the mobile phase at elution (ke) [START_REF] Sarrut | Optimization of conditions in on-line comprehensive two-dimensional reversed phase liquid chromatography. Experimental comparison with one-dimensional reversed phase liquid chromatography for the separation of peptides[END_REF][START_REF] Chapel | A Theoretical and Practical Approach to Manage High Peak Capacity and Low Dilution in On-line Comprehensive Reversed-Phase LC × Reversed-Phase LC: A Comparison with 1D-Reversed-Phase LC[END_REF][START_REF] Groskreutz | Quantitative evaluation of models for solvent-based, oncolumn focusing in liquid chromatography[END_REF]:

L = M N M F (8)
For this reason, the difference, ∆C, between the composition of the injection solvent and the composition at peak elution, can be considered as a measure of the solvent strength mismatch:

∆ = D E - (9)
Solvent strength mismatch is favourable when ∆ < 1. It is all the more critical as ∆C is much higher than 1.

With ASM, the duration of the dilution step and hence the required dilution volume must be specified. This latter must be at least equal to < D E . However, a larger volume, expressed as a multiple,

λ D E , of < D E (with λ D E > 1
) is recommended to ensure the complete dilution of the fraction stored in the loop. It should be noted that the settable parameter required by the software is the number of loop volumes (λ 7 ) in place of the number of injection volumes (λ D E ). It can be derived from the desired λ D E by:

λ 7 = λ D E × L × R 'IIS (10)
A value of 3 is recommended for λ 7 by Agilent. However, this value is most often too high considering the very short cycle times needed in sub-hour on-line LC x LC. To dilute the injection plug with the initial mobile phase, an initial isocratic hold has to be included in the pump program so that the gradient starts after the dilution step. Its duration, 2 tiso, is recommended to be the same as that of the dilution step and hence given by:

8 = λ $%TFU)$I% × R $%TFU)$I% L (11)
The analysis time in 2 D using ASMS includes this initial hold and consequently, the available gradient time is decreased compared to Eq.4 according to:

As a rule of thumb, the volume occupied by the fraction in the loop, < , E , should not exceed two-thirds of the loop volume (< 7 ≥ 1.5 × < , E ) to take into account the molecular dispersion due to the parabolic flow. With ASMS, because the dilution is achieved after the sample loop, < , E is the same as with TBS (?. X. C = × ) whereas with FSS and MFS, it corresponds to the injection volume (Eq. 6). Whereas small loop volumes can be used with FSS (zsplit < 1) or even with TBS or ASMS, larger ones are required with MFS (zdilution > 1). Accordingly, the available time for the gradient is shorter (Fig. 2). Of greater practical consequence is the fact that the peak capacity is expected to be lower since the peak capacity decreases with the gradient time.

The interface conditions for the four different strategies and the remaining gradient times are listed in Table 1. For a fair comparison, the chromatographic conditions were basically the same and quite close to those previously applied to the on-line HILIC x RPLC analysis of a tryptic digest [START_REF] Chapel | Pushing the limits of resolving power and analysis time in on-line comprehensive hydrophilic interaction x reversed phase liquid chromatography for the analysis of complex peptide samples[END_REF]. The gradient time in 1 D (assumed to be the analysis time) was 30 min. The analysis time in 2 D (i.e. the sampling time) was 0.39 min. The flow-rate in 2 D (i.e. 2 mL/min) was fixed in such a way that the maximum allowable pressure (i.e. 1000 bar) could be reached. The sample loop volumes were selected among commercially available loops so that the injection volume did not exceed two-thirds of the loop volume. As shown in Fig. 2, some times during the run in 2 D were maintained identical for the four studied strategies. Those include 2 tD,instrum, 2 tpost, and 2 teq equal to 0.022, 0.030 and 0.058 min, respectively. Accordingly, interface conditions being different, the gradient times in 2 D (Table 1)

were also different depending on the strategy (see Eqs. 4 and 10).

With TBS (Figs. 1a and2), no additional device is required, making this strategy very easy to operate.

The whole fraction coming from 1 D was injected in 2 D. That resulted in relatively large volumes of strong solvent injected in 2 D. For the separation of peptides, the percentage of ACN in the HILIC fractions was between 98% and 48% depending on the solute retention in HILIC. A conventional twoposition/4-port duo-valve was used in the backflush mode (Fig. S1). Considering the flow rate in 1 D (i.e. 50 µL/min) and the sampling time of 0.39 min, the volume of each transferred fraction and hence the volume injected in 2 D, was exactly 19.5 µL. That represented about 27% 2 V0 ( 2 V0 being the 2 D-column dead volume). Considering commercially available loop sizes and with respect to a filling percentage of no more than 66%, the selected sample loop volume was 40 µL. Accordingly, 2 tloop was 0.02 min, resulting in a gradient time of 0.26 min (Eq. 4).

With FSS (Figs. 1b and2), restriction capillaries were mounted to provide a split 1:9 (zsplit = 0.1), thus allowing sending nine-tenths of the flow to the waste while one-tenth to the sample loop. As said above, a split ratio of 1/10 with similar inner diameters (I.D) in both dimensions (e.g.. 2.1 mm) mimics a situation in which the column I.D ratio would be close to 3 (e.g. 1 mm in 1 D and 3 mm in 2 D). Under these conditions, the injected volume in the second dimension was exactly 1.95 µL. That represents about 2.7% V0. By reducing the loop volume down to the lowest available standardized loop volume (i.e. 20 µL), the gain in time was rather small (of the order of 0.01 min) but allowed for a very small increase in the gradient time (0.27 min).

With both MFS and ASMS, a dilution ratio of 5 (maximum dilution ratio with ASMS) was selected.

This one was recommended in case of strong solvent strength mismatch [START_REF] Stoll | Active Solvent Modulation: A Valve-Based Approach To Improve Separation Compatibility in Two-Dimensional Liquid Chromatography[END_REF]. A lower dilution ratio would reduce the transfer volume (Eq. 6) but also increase the injection solvent strength (Eq. 7).

With MFS (Figs. 1c and2) and a dilution ratio of five, Fpump was 200 µL/min (Eq.2). The diluting solvent composition was the same as for the starting composition of the 2 D mobile phase (i.e. 1% ACN + 0.1% FA and 99% water + 0.1% FA). In these conditions, the percentage of ACN in the injection solvent was decreased from 98% to 20.4% in 1 D initial conditions and from 52% to 11.2% in 1 Considering the sample loop volume, twice as large as the fraction volume, only one sample loop volume (λloop = 1 and λinjection close to 2) was used to flush the loop. As a result and according to Eq. 11, a dilution step and hence an isocratic hold ( 2 tiso) of 0.1 min was required for complete sample dilution which, according to Eq. 12, reduced the available gradient time by about 16% compared to MFS (0.16 min vs. 0.19 min).

On-line HILIC x RPLC with TBS or FSS

The main difference between FSS and TBS is the use of restriction capillaries with FSS which divides the flow before entering the valve. This results in lower injection volumes (here ten-fold lower than with TBS). With TBS, as discussed above, the injection volumes in 2 D are large enough to attain a situation of Total Breakthrough for all peptides. As previously defined [START_REF] Chapel | Pushing the limits of resolving power and analysis time in on-line comprehensive hydrophilic interaction x reversed phase liquid chromatography for the analysis of complex peptide samples[END_REF], Total Breakthrough exists when there are only two distinct peaks for a given solute, the first one unretained (breakthrough) and the second one retained and quite symmetrical. It was found for peptides that Total Breakthrough occurs when the injection volume is higher than a critical one. For smaller volumes, a transition step was pointed out, in which breakthrough occurs while the retained peak is distorted and sometimes split. With FSS, despite a much lower injection volume (1.95 µL vs. 19.5 µL), some peaks of breakthrough are still present, although less intense than with TBS (Fig. 3b vs. Fig. 3a). This is not surprising since breakthrough has been observed for peptides in on-line HILIC x RPLC with injection volumes as low as 2% V0 [START_REF] Vanhoenacker | Comprehensive twodimensional liquid chromatography of therapeutic monoclonal antibody digests[END_REF]. Breakthrough could have been mitigated provided that lower volumes had been injected in 2 D. In our case, this could have been done by means of larger split ratios between the two dimensions (e.g. 1/26 for Vi = 1% V0). Nevertheless, we do not believe that breakthrough could have been eliminated given that its occurrence was reported with injected volumes as low as 0.5% V0 under similar chromatographic conditions [START_REF] Chapel | Pushing the limits of resolving power and analysis time in on-line comprehensive hydrophilic interaction x reversed phase liquid chromatography for the analysis of complex peptide samples[END_REF]. Furthermore, such a decrease in injection volume would have significantly impacted the method sensitivity, as will be underlined in the subsequent section.

As shown by dotted lines in Fig. 3, the entire separation space is occupied by peaks in HILIC x RPLC (i.e. γ = 1 in Eq.1). Key values for the effective peak capacity calculation (Eq. 1) are listed in Table 2.

The average peak widths in 1 D and 2 D (UV detection) were assessed from about fifty single symmetrical peaks. The conditions in 1 D being the same with the four strategies, 1 ∆t and 1 w4σ values are the same. 2 ∆t is slightly larger with FSS but the main difference comes from average 2 w4σ values (0.39 s with TBS vs. 0.46 s with FSS), resulting in a higher peak capacity (i.e. 1100 with TBS vs. 970 with FSS). As discussed before, TBS ensures a situation of Total Breakthrough due to an injection volume well above the critical one for all peptides. With FSS, the injection volume is most time too low for Total Breakthrough but not low enough to avoid broadening, distortion or breakthrough, as will be further discussed. In addition to the effect of injection solvent strength, low split ratios (here zsplit = 0.1) can also significantly broaden the peaks [START_REF] Bernardin | Suitable interface for coupling liquid chromatography to inductively coupled plasmamass spectrometry for the analysis of organic matrices. 1 Theoretical and experimental considerations on solute dispersion[END_REF]. It is important to note that peak widths could only be measured on symmetrical peaks which makes the peak capacity of 970 very optimistic.

3D-chromatograms bring attention to the significant difference in peak intensity between TBS (Fig. S5a) and FSS (Fig. S5b). This difference can also be assessed with the overlaid 2 D-chromatograms shown in Fig. S6. With a view to even better assess the difference in peak intensity between the studied strategies, 39 different single peaks, identified by HRMS, were selected. Those peaks had to be well distributed among the whole retention space with a signal-to-noise ratio higher than five (UV detection) for accurate measurements. Their peak height ratios between TBS and FSS are listed in Table 3. The largest peak height ratios concerned the most retained peaks in 2 D. Their intensities were less affected since the intensity of the peak of breakthrough decreased as the peptide retention increased as earlier discussed [START_REF] Chapel | Pushing the limits of resolving power and analysis time in on-line comprehensive hydrophilic interaction x reversed phase liquid chromatography for the analysis of complex peptide samples[END_REF]. On average, the peak intensity was found to be about 8-fold higher with TBS, ranging from 3-to 22-fold. As seen in Table 2, about 99% of the expected peptides from the trypsin digestion (i.e. 237/238, including known post-translational modifications)

were identified with HRMS detection for TBS. In comparison, only 86% (i.e. 205/238) could be unambiguously identified for FSS due to low detection sensitivity. These results underline the high dilution caused by FSS conditions.

Finally, considering both peak capacity (Table 2) and peak intensity (Table 3), TBS was proved to be a much better choice than FSS to reduce undesirable effects of strong injection solvents in the 2 D-RPLC.

On-line HILIC x RPLC with MFS or ASMS

Both strategies consist in diluting the injection plug with a weak solvent (here 1% ACN in water with 0.1% FA). The eluent strength of the 1 D-effluent is therefore decreased before entering the 2 Dcolumn to promote column focusing. On-line dilution was achieved before the switching valve (MFS)

or inside the switching valve (ASMS). The contour plots and the 3D-chromatograms for both MFS and ASMS are shown in Fig. 3 and Fig. S5, respectively. The similarity between the two separations is noteworthy. As a consequence of the large isocratic hold before the gradient enters the column, a large empty space with MFS (Fig. 3c) and even larger with ASMS (Fig. 3d) is clearly observed. Since the sampling time was identical for all strategies, the available gradient time (Fig. 2) and hence the separation space (i.e. 2 Δt in Table 2) was inevitably much smaller compared to TBS or FSS (27% and 40% smaller with MFS and ASMS, respectively). As could be expected, a smaller average peak width was obtained with both MFS and ASMS compared to FSS (Table 2). The average peak width was yet close to that with TBS. Due to the shorter 2 Δt with both dilution approaches, peak capacities were much smaller with MFS (770) and ASMS (680) than with TBS (1100) or even FSS (970). Furthermore, despite on-line dilution, intense peaks of breakthrough are still observed with MFS as well as with ASMS (Fig. 3), especially for moderately retained compounds in 1 D.

As seen in Fig. S5 and confirmed by Table 3, the peak heights of the retained peaks are similar between MFS and ASMS. On average, they were about five times higher than with TBS. The presence of breakthrough did not seem to impair much the peak intensity. As seen in Table 2, the percentage of expected peptides identified with both strategies was the same as for TBS.

In the next section, the comparison of the peak shapes from HRMS detection will complete the evaluation of the four strategies. are ranged from the most retained peptide in 2 D (Fig. 4) to the least retained one (Fig. 8). The location of each peak is indicated on the 2D-maps in Fig. S4. Useful information, listed in Table 4, includes m/z values, retention times in both dimensions, compositions at elution in both dimensions (Eq. 2), compositions of the injection solvent in 2 D (Eq. 7), measured effective peak capacities (Eq. 1), observed type of separation (Total Breakthrough, single symmetrical peak, peak distortion, split peak, transition step accompanied by both peak distortion and breakthrough) and peak asymmetry factors (Eq. 3). Note that the effective peak capacities, as well as the asymmetry factors, could not be measured when the peak distortion was significant or when the peak intensity was too low. It should be pointed out that the effective peak capacities were calculated from MS-peak widths in Table 4 but from UV-peak widths in Table 2. The resulting lower values are due to significant additional solute dispersion between the UV-instrument and the Q-TOF-MS connected in parallel with a tee-piece.

Finally as previously said, ∆C (Eq. 9) can be considered as a measure of the solvent strength mismatch and was therefore also reported in Table 4 as additional relevant characteristic.

The peptide in Fig. 4 is strongly retained in both dimensions. As a result, the injection solvent strength is moderate (57% and 54%ACN with TBS and FSS respectively) and more importantly, ∆C is low with both TBS and FSS (i.e. 25%) and negative with both MFS and ASMS (i.e. -20%). As expected, a small peak of breakthrough appears with TBS. In such conditions, the retained peak is kept rather symmetrical with all strategies (i.e. 1 ≤ As ≤ 1.6, except for ASMS). However, for the reasons already given above, the effective peak capacities are much lower with MFS and ASMS.

In Figs. 5 to 7, the peptides are poorly retained in 1 D ( 1 Celution from 86% to 71%ACN) while moderately retained in 2 D ( 2 Celution from 29% to 15% ACN), which results in high ∆C values with TBS and FSS (about 60% ACN). With FSS, despite a very small injection volume, the peaks are split in Figs. 5b and 6b while Total Breakthrough similar to what is always observed with TBS can be observed in Fig. 7b.

Regarding MFS and ASMS, the ∆C values remain negative for the peptide in Fig. 5 (i.e. close to -10%), thus allowing a fairly symmetrical retained peak. However, ∆C values seem to be too high for the peptides in Figs. 6 and7, taking also into account the very large injection volume resulting from these strategies (i.e. 97.5µL). Peak fronting can be observed with MFS in Fig. 6c and a peak of breakthrough appears with ASMS in Fig. 6d, suggesting a transition step before a further situation of Total Breakthrough. The earlier emergence of breakthrough in the case of ASMS can be readily explained by the slight difference in ∆C between the two strategies. In Fig. 7, the peaks are distorted with both MFS (Fig. 7c) and ASMS (Fig. 7d). In addition to the very bad peak shapes, a peak of breakthrough is present in both cases. This suggests that on-line dilution is not sufficient to circumvent the deleterious impact of solvent strength mismatch and thus to ensure symmetrical peaks. This bad separation can be related to the ∆C values (here close to 0%), higher than for the peptide in Fig. 6 and much higher than for the peptide in Fig. 5.

Three approaches could be considered to reduce the transferred volume while maintaining the same dilution ratio (same ∆C): (i) splitting the flow-rate prior to the valve, (ii) sending part of the fraction to the waste by using loop volumes smaller than the fraction volume or (iii) using trapping columns.

The first approach involves the combination of two strategies (FSS and on-line dilution). The second one could not strictly refer to as comprehensive 2D-LC and furthermore cannot be considered for quantitative analysis since the percentage of component sent in the second dimension may vary from run-to-run. Furthermore, these two approaches should result in a reduction of the peak heights (lower sensitivity). Regarding the use of trapping columns in combination with on-line dilution (MFS only), its benefit in terms of transferred volume reduction has never been clearly proved as above highlighted.

The last proposed example (Fig. 8) is interesting. In this case, the peptide is strongly retained in 1 D like the peptide in Fig. 6 ( 1 Celution close to 60% ACN) but poorly retained in 2 D ( 2 Celution close to 6% ACN). With both TBS and FSS (Figs. 8a and8b), the situation of total breakthrough is attained. With MFS and ASMS (Figs. 8c and8d), a transition step close to a situation of Total Breakthrough but with a low fronting between the peak of breakthrough and the retained peak can be observed. Once again, these results can be correlated to the high ∆C values (here positive values close to 7% ACN). It is interesting to point out that the breakthrough peak is much larger with MFS (Fig. 8c) and ASMS (Fig. 8d) than with TBS (Fig. 8a). It is the thinnest with FSS (8b). That is not surprising considering that the width of the non-retained breakthrough peak is expected to vary linearly with the injected volume.

Apart from obvious consequences on separation and sensitivity, the occurrence of breakthrough in each of these strategies raises the question of quantitative analysis. In our previous study [START_REF] Chapel | Pushing the limits of resolving power and analysis time in on-line comprehensive hydrophilic interaction x reversed phase liquid chromatography for the analysis of complex peptide samples[END_REF], we observed a linear variation of the retained peak area with the amount of peptide injected, as soon as the situation of Total Breakthrough was attained. However, the variation was no more linear when the peak was eluted under the conditions of transition step (i.e. after the emergence of breakthrough but before Total Breakthrough). This suggests that quantitative analysis should be quite reliable with TBS while problematic with the three other strategies (transition step with FSS in Figs. 6b, with MFS in Fig. 7c or with ASMS in Fig. 7d).

In summary, the following conclusions can be drawn from the separations shown in Figs. 4 to 8: (i)

TBS was the only strategy ensuring fairly symmetrical peaks (1 ≤ As ≤ 1.6) -for all peptides in on-line HILIC x RPLC, (ii) the success of the three other strategies (FSS, MFS, and ASMS), for a given peptide, strongly depended on ∆C values which had to be low enough to avoid injection issues, (iii) considering that it was not possible to ensure sufficiently low ∆C values for all peptides given the broad range of elution in both dimensions, the peak capacity and hence the quality of the separation was inevitably more or less affected with MFS and ASMS, and (iv) MFS and ASMS yielded quite similar results with the same dilution ratio.

In this study, TBS was used for the separation of peptides. We are currently studying the occurrence of Total Breakthrough over a wider range of solutes (acidic and basic, multi-charged or not) and a wider range of chromatographic conditions (different stationary phases, different mobile phases, pHs, temperatures, gradient conditions). Our obtained results allow asserting that Total Breakthrough can also be attained with small monocharged molecules in RPLC. An example is given in figure S7 for propranolol. We hope to be able soon to publish a complete overview of the circumstances leading to the phenomenon of Total Breakthrough.

Concluding remarks

In this study, we compared four strategies designed to reduce undesirable effects of strong injection solvents on peak shapes in the second dimension of on-line HILIC x RPLC for the separation of peptides: (i) the Total Breakthrough strategy (TBS) with injection of large volumes of raw fractions, (ii) the flow splitting strategy (FSS) with injection of low volumes of raw fractions, and two on-line dilution strategies with injection of very large volumes of diluted fractions, either (iii) with a make-up flow (MFS) or (iv) with an ASM-valve (ASMS). The quality of the separation was assessed on the basis of peak shapes, peak capacity, and peak intensity. The comparison was made in the context of a separation of a complex peptide sample performed in 30 min by considering the most critical case where similar column inner diameters are used in both dimensions (i.e. 2.1 mm).

The best results in term of peak shapes and peak capacity were obtained with TBS. A feature of this strategy is that it deliberately copes with breakthrough, unlike the three other ones where breakthrough is highly critical. No additional device is required with TBS, making this strategy very easy to operate. A peak capacity of 1100 could be achieved within just half an hour. Due to both peak broadening and a reduction of the separation space in 2 D, the obtained peak capacity was more than 40% lower with the on-line dilution strategies. Unlike TBS, it was shown that neither a split ratio of 1/10 (FSS) nor a dilution ratio of 5 (MFS and ASMS) were sufficient to provide symmetrical peaks across the entire 2D-separation space and hence to avoid excessive band broadening, fronting, split peaks, and breakthrough.

The difference in composition between the injection solvent and the mobile phase at peak elution (∆C) in combination with the injection volume was found to be related to the extent to which peaks are distorted and also to the occurrence of breakthrough. Above a critical ∆C value depending on the injection volume, a situation of Total Breakthrough takes place. Due to low ∆C values, this situation was rarely attained with MFS or with ASMS.

On average the peak intensity was five times higher with the on-line dilution strategies than with TBS. However, TBS led to much better results than FSS, with an eight-fold increase in peak intensity.

This highlights the poor sensitivity involved by FSS while this strategy is still the most reported one in on-line HILIC x RPLC.

ASMS and MFS gave very similar results. Both techniques need an additional device, either an ASM valve or an isocratic pump (MFS). This latter solution benefits from easy implementation.

Furthermore, the present study shows that the current limitation for the dilution ratio (not higher than 5) in the case of the ASM valve can be a critical issue for the quality of the separation in on-line HILIC x RPLC.

Finally, there are many ways to compare the different strategies for given 1 D conditions. The quality descriptors are multiple (peak height, peak width, peak capacity, peak asymmetry…).

Depending on the objectives, one of the descriptors may prevail over another. However peak heights, peak widths and hence peak capacity cannot be assessed if the peaks are distorted. The first objective of the analyst should therefore be to obtain only quasisymmetrical peaks. The main conclusion of this study on HILIC x RPLC is that this objective can only be achieved with TBS. 1. Other conditions given in experimental section and in Table 1. 4. 4. 4. 4.

Fig. 3

 3 Fig. 3 shows the 2D-contour plots obtained for the separations of the tryptic digest with TBS (Figs. 3a) and FSS (Figs. 3b), with a view centred on the useful separation space. The entire 2D-contour plots are shown in Fig. S4. The 3D-plots are given in Fig. S5 to compare the peak intensities of the two separations. With TBS, an intense and large band appears between 2 and 3 seconds. It corresponds to the expected 2 D-column dead time (about 2.2 s) and is therefore composed of peaks of breakthrough as also confirmed by HRMS. Those are omnipresent in 2 D during the entire 1 D separation. As can be observed, they do not enter the separation space delimited by dotted lines.

3. 4 .

 4 Comparison of peak shapes in on-line HILIC x RPLC with the four studied strategies To provide an objective comparison of the peak shapes depending on the studied strategies, extracted ion chromatograms (EICs) of five relevant peptides are shown in Figs. 4 to 8. Each separation displays the most intense fraction among the two or three 1 D-peak fractions. The figures

Figure captions Figure 1 :Figure 2 :

 captions12 Figure captions

Figure 3 :

 3 Figure 3: On-line HILIC x RPLC separations (2D-contour plots) of a tryptic digest of 6 proteins with (a) Total breakthrough strategy (TBS), (b) Flow splitting strategy (FSS), (c) Make-up flow Strategy (MFS), and (d) Active solvent modulation strategy (ASMS). The white dotted lines delimit the separation space. UV detection at 210 nm. Other conditions given in experimental section and in Table1.

Figure 4 :

 4 Figure 4: 2 D-separation of a strongly retained peptide (EIC 1622.5389) with four studied strategies used in on-line HILIC x RPLC: (a) Total breakthrough strategy (TBS); (b) Flow splitting strategy (FSS), (c) Make-up flow Strategy (MFS), and (d) Active solvent modulation strategy (ASMS). The characteristics of the retained peak are given in Table4.

Figure 5 :

 5 Figure 5: 2 D separation of a moderately retained peptide (EIC 588.3769) with four studied strategies used in on-line HILIC x RPLC: (a) Total breakthrough strategy (TBS); (b) Flow splitting strategy (FSS), (c) Make-up flow Strategy (MFS), and (d) Active solvent modulation strategy (ASMS). The characteristics of the retained peak are given in Table4.

Figure 6 :

 6 Figure 6: 2 D separation of a moderately retained peptide (EIC 723.3568) with four studied strategies used in on-line HILIC x RPLC: (a) Total breakthrough strategy (TBS); (b) Flow splitting strategy (FSS), (c) Make-up flow Strategy (MFS), and (d) Active solvent modulation strategy (ASMS). The characteristics of the retained peak are given in Table4.

Figure 7 :

 7 Figure 7: 2 D separation of a moderately retained peptide (EIC 779.4492) with four studied strategies used in on-line HILIC x RPLC: (a) Total breakthrough strategy (TBS); (b) Flow splitting strategy (FSS), (c) Make-up flow Strategy (MFS), and (d) Active solvent modulation strategy (ASMS). The characteristics of the retained peak are given in Table4.

Figure 8 :

 8 Figure 8: 2 D separation of a poorly retained peptide (EIC 830.4534) with four studied strategies used in on-line HILIC x RPLC: (a) Total breakthrough strategy (TBS); (b) Flow splitting strategy (FSS), (c) Make-up flow Strategy (MFS), and (d) Active solvent modulation strategy (ASMS). The characteristics of the retained peak are given in Table4.

  ).2 tpost is the short time required for going back to the initial composition and 2 teq, the column equilibration time. Both 2 tpost and 2 teq can be expressed as multiple of the column dead time ( 2 t0), typically one 2 t0

	instrument dwell time without considering the sample loop (	=	7 +	, 8 , 9
	5 =	-6	7 +	, 8 , 9 + 7 8 +	: ;		(4)

7 is the time required for the 2 D-mobile phase to travel across the sample loop ( 7 = < 7 = , > ? ℎ < 7 the loop volume and = , the flow-rate in 2 D). 2 tD,instrum is the

Table 1 :

 1 Experimental interface conditions for the four compared strategies: total breakthrough strategy (TBS), flow splitting strategy (FSS), on-line dilution with make-up flow strategy (MFS), and on-line dilution with ASM strategy (ASMS). The different terms refer to Eqs. 3 to 9. 2 F = 2000 µL/min; sampling time = 0.39 min. The graphical representation of the time repartition is presented in Fig.3.

		1 Vfraction (µL)	1 zsplit	1 zdilution	Vloop (µL)	2 Vinjection (µL)	2 tloop (min)	2 tD,instru (min)	2 tiso (min)	2 tG (min)	2 tpost (min)	2 teq (min)
	TBS	19.5	1	1	40	19.5	0.02	0.04	0	0.26	0.03	0.04
	FSS	1.95	10	1	20	1.95	0.01	0.04	0	0.27	0.03	0.04
	MFS	97.5	1	5	180	97.5	0.09	0.04	0	0.19	0.03	0.04
	ASMS	19.5	1	1	40	97.5	0.02	0.04	0.1	0.16	0.03	0.04

Table 2 :

 2 Effective peak capacities (n2D,eff) and number of peptides identified compared to the expected number of peptides with the four compared strategies: Total Breakthrough strategy (TBS), Flow splitting strategy (FSS), On-line dilution with make-up flow strategy (MFS), and on-line dilution with ASM strategy (ASMS). Intermediate parameters for the peak capacity calculation (Eq. 1): ranges of elution times (Δt) and average 4σ peak widths (w4σ) measured with UV detection (210 nm). Other values required in Eq.1: α = 0.673 and γ = 1.

		1 D		2 D			Number of
		1 ΔT (min)	1 w 4σ,UV (min)	2 ΔT (s)	2 w 4σ,UV (s)	n2D,eff (UV)	peptides identified (%)
	TBS			15.0	0.39	1100	99
	FSS			15.6	0.46	970	86
	MFS	26	0.65	11.0	0.41	770	99
	ASMS			9.0	0.38	680	99

Table 3 :

 3 Peak height ratios between different strategies for 39 relevant peaks well spread among the 2D-retention space. UV detection at 210 nm.

	Peak #	1 t r	m/z	Peak height ratio TBS/FSS MFS/TBS ASMS/TBS MFS/ASMS
	1	21.1 1156.9329	22	1.1	3.5	0.3
	2	21.4	1368.221	20	1.2	3.3	0.4
	3	19.9	742.4514	4.1	6.0	5.8	1.0
	4	26.9 1557.3634	9.6	3.1	2.6	1.2
	5	30.4 1622.2051	21	3.7	3.3	1.1
	6	19.9 1300.6981	5.3	6.8	6.8	1.0
	7	23.8	864.9531	4.7	4.2	4.0	1.1
	8	33.9 1057.5826	21	3.4	2.8	1.2
	9	19.9 1231.5983	8.4	5.8	3.3	1.7
		23.0	908.9567	6.1	6.9	6.4	1.1
		26.1	680.9209	9.0	3.1	3.3	0.9
		28.5	761.3974	7.6	4.8	6.2	0.8
		30.8	460.2878	6.9	4.0	4.5	0.9
		24.2	682.7068	6.6	3.5	4.5	0.8
		25.0	748.3533	4.2	5.6	7.1	0.8
		20.3	478.2555	6.3	7.1	5.6	1.3
		22.6	482.772	8.0	13	13	1
		23.0	692.4062	8.3	9.1	8.3	1.1

Acknowledgements

The authors gratefully acknowledge Agilent technologies for the loan of the 2D-LC instrument and the Active Solvent Modulation 2D-LC valve. (a) TBS (b) FSS (c) MFS (d) ASMS (a) TBS (b) FSS (c) MFS (d) ASMS (a) TBS (b) FSS (c) MFS (d) ASMS (a) TBS (b) FSS (c) MFS (d) ASMS (a) TBS (b) FSS (c) MFS (d) ASMS