Radical Coupling Allows a Fast and Tuned Synthesis of Densely

Article pubs.acs.org/Macromolecules

Radical Coupling Allows a Fast and Tuned Synthesis of Densely Packed Polyrotaxanes Involving γ‑Cyclodextrins and Polydimethylsiloxane F. Blin,† C. Przybylski,† V. Bonnet,§ M. J. Clément,‡ P. A. Curmi,‡ P. Choppinet,∥ T. Nakajima,∥ H. Chéradame,† and N. Jarroux*,† †

Laboratoire Analyse et Modélisation pour la Biologie et l’Environnement, CNRS UMR 8587, and ‡Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, INSERM UMR 829, Université d’Evry-Val d’Essonne, Bâtiment Maupertuis, bd F. Mitterrand, 91025 Evry, France § Laboratoire de Glycochimie, des Antimicrobiens et des Agroressources, CNRS UMR 7378, Université de Picardie Jules Verne, 33 rue Saint-Leu, 80000 Amiens, France ∥ Applied Technology Development Department, Menicon Co., Ltd., Kasugai 4870032, Japan S Supporting Information *

ABSTRACT: The first radical end-coupling synthesis of polydimethylsiloxane (PDMS)−γ-cyclodextrins (γ-CDs) based polyrotaxane is reported. Conversely to usual chemical way, the radical process leads to fast both controlled size and structure with minimal side reaction while exhibiting very high conversion rate (w/w, 80%). Pure PDMS−γ-CDs molecular necklaces were successfully isolated by preparative size exclusion chromatography and finely characterized both by 1D/2D/STD 1H and 13C NMR and MALDI-TOF mass spectrometry. The observations give clear evidence of the supramolecular assembly synthesis where the filling ratio (γ-CD/monomer unit) of PDMS chains is as high as 40% of γ-CDs. Combination of such radical-based coupling supported by detailed analytical characterizations appears at the forefront of a fast, suitable, and easily amenable scaling-up CDs-based polyrotaxane synthesis process.

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INTRODUCTION Polyrotaxanes (PR) are interlocked macromolecule composed of macrocyclic compounds threaded onto a polymer chain, where the two extremities are blocked with bulky groups to avoid dethreading.1−6 These supramolecular entities have gained considerable attention because the mobility of cyclic molecules could lead to unique properties.7,8 Cyclodextrins (CDs) are cyclic oligosaccharides constituted of 6, 7, or 8 glucopyranoside units (α-, β-, and γ-, respectively) linked by α(1 → 4) bonds, which present several advantages such as the presence of multiple functionalizing groups, a relatively low cost, and an intrinsic low toxicity.9,10 For all these reasons, CDs are macrocycles of choice for numerous PR, as reported in several excellent reviews dealing with both their synthesis and applications.11−18 Many different linear polymers have been used to construct new PR with α-, β-, or γ-CDs.11,13,15 In 1990, Harada et al. introduced this type of supramolecular structure by threading α-CD onto poly(ethylene glycol) (PEG) polymer backbone.19 It was quoted out that the efficiency of a PR synthesis is strongly affected by the nature and the molecular weight of the polymer and the choice of the macrocycle. The average filling ratio obtained is also a critical factor which directly affects the properties of the PR, thus governing its potential applications. © XXXX American Chemical Society

Among the various polymers usually used, polydimethylsiloxane (PDMS) presents very attractive properties due to its unique rheological behavior, intrinsic flexibility, and thermostability. These features offer the possibility to use this material in many different fields and to develop PDMS-based PR as a new class of organic−inorganic materials,20 which is still largely under investigation. In 2000, Harada et al. reported for the first time the formation of PDMS inclusion complexes with β-CD and γ-CD.21,22 Nevertheless, to the best of our knowledge, only two groups effectively reported the coupling reaction of α,ωtelechelic PDMS with specific blocking groups to avoid the dethreading of the γ-CDs.23−26 Compared to wider synthesized PEO-based PR, using PDMS dramatically changes the synthesis conditions leading to the formation of PR.24 In such study, it was reported that the low both conversion yield from PPR to PR and filling ratio for the PDMS-based PR were due to (i) intrinsic hydrophobic nature of the PDMS polymer chain, (ii) extremely poor accessibility of the PDMS telechelic functions to the capping agents (difference of solubility between PDMS, PDMS-based PPR, and the capping agents), and (iii) PDMS’s Received: March 9, 2016 Revised: April 12, 2016

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DOI: 10.1021/acs.macromol.6b00492 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules

Table 1. Impact of the Reaction Medium Composition on Conversion Yield from Pseudopolyrotaxane to Polyrotaxane Synthesis with Initiatior (Na2S2O8) at 5% (w/w) run no.

[PPR]/[PBS] (mol/mol)a

additional [γ-CD]/[pyrene] (mM/mM)

PR in crude productb (%)

PR in crude productc (w/w, %)

PR conv yieldd (w/w, %)

cosolvent

1 2 3 4 5

10/100 10/100 10/100 2/20 2/20

0/0 0/0 180/0 180/0 180/180

2 15 14 25 24

5 13 19 25 23

2 14 27 78 81

without Et2O Et2O Et2O Et2O

a

M̅ n of the PPR was estimated to be equivalent to the full packed PPR.21,22 bThe percentage of PR was estimated by SEC with comparison with all area of detected peaks. cThe percentage of PR was estimated by SEC of the crude product after calibration and quantification of all residues present in the sample. dThe polyrotaxane conversion yield was estimated according to the equation (w/w, %) yield = % PR × (weight of crude product/ weight of PPR).

“thicker” main chain polymers which requires larger CDs (βCD or γ-CD), where (i), (ii), and (iii) favor the dethreading of the macrocyle during PPR formation. The main consequence is that synthesis of PDMS-PR is sparsely populated with γ-CD, reaching a filling ratio equal to 7%. Modification of the hydroxyl groups of the threaded γ-CD by acetylation or silylation improved the difference of solubility between free and threaded CDs in various solvents, such as chloroform, dichloromethane, and THF, enhancing the purification step efficiency. Effective rotaxanation was finally proved by size exclusion chromatography (SEC) but with moderate yield (53%), constituting however the first organic−inorganic hybrid slide-ring gel.24 Obviously, dethreading of the macrocycles during synthesis can become an issue when trying to prepare densely packed PDMSPR. Unfortunately, a straightforward method to accurately control the effectiveness of such polyrotaxane synthesis with a finely tune of the filling ratio was not yet been found. One primary reason is that specific end-capping synthesis methodologies need to be developed. A second likely cause hold in the absence of suitable method to purify the final products independently of the number of CDs threaded into the polymer chain, especially regarding the presence of remaining free macrocycles. In this sense, some authors Simionescu et al. stated that the PR synthesis based on PDMS and γ-CD cannot lead to pure macromolecular edifice when the polymer chain is densely packed with macrocycles.24 This can be explained by the fact that the solution behavior of a macromolecular assembly containing a large amount of CDs is close of free CDs. This phenomenon is also presumably exacerbated by a non-negligible hydrogen bonds network between free and threaded CDs. Such a side effect could be eliminated or strongly reduced by a judicious modification of the hydroxyl groups of the CDs. All these bottleneck can, in part, explain both why obtaining PDMS-based PR with tuned filling ratio higher than 7% was not yet been achieved and why a good purification and a fine structural characterization remain challenging tasks. The present study reports efforts to tackle these bottlenecks using a newly introduced synthetic chemistry, the radical reaction, to block polymer chain ends during conversion of PDMS-based pseudopolyrotaxane (PPR) to PR. The radical coupling method owns a usefulness which has been already demonstrated to obtain fully packed PEO based PR in a very high yield (96%).27 Radical end-coupling enables a fast and ecofriendly synthesis route to obtain high and tuned filling ratio of the macromolecular structures. Such an original approach greatly helps to develop a suitable SEC purification step yielding to pure PR and then opening the way to an accurate

characterization both by usual 2D 1H/13C NMR and by a dedicated MALDI-TOF mass spectrometry (MALDI-TOF MS).

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EXPERIMENTAL SECTION

Materials. γ-Cyclodextrin (γ-CD) was supplied by Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan). They were used after drying under vacuum for 16 h at room temperature. α,ωDimethacrylate polydimethylsiloxane (α,ω-dimethacrylate PDMS, 4600 g mol−1) was kindly provided by Shin-Etsu Chemical Co. Ltd. (Tokyo, Japan). The average molecular weight of polymer samples and the methacrylic functionality ( f = 2) were checked by 1H NMR. Sodium persulfate used as radical initiator, sinapinic acid (SA), 2′,4′,6′trihydroxyacetophenone (THAP), 1,1,3,3-tetramethylguanidine (TMG), lithium chloride (LiCl), and 1-pyrenebutyric acid Nhydroxysuccinimide ester (PBS) used as blocking agent were purchased from Sigma-Aldrich Co. (St Quentin Fallavier, France) and used as received. Ultrapure water was prepared by passing distilled water through a Quantum Ultrapure Organex cartridge (QTUM000EX, Millipore). Dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and diethyl ether (Et2O) were purchased from SDS Carlo Erba (Val de Reuil, France) and used as received. Preparation of the Inclusion Complexes: Pseudopolyrotaxane PDMS/γ-CD. A saturated aqueous solution of γ-cyclodextrins (10 g in 43 mL) was poured into a 100 mL round flask containing α,ωdimethacrylate polydimethylsiloxane (0.921 g). According to Harada’s findings, one γ-CD cavity can accommodate 1.5 siloxane units (3nSi(CH3)2-O = 2nγ-CD);22 at this stoichiometry, the γ-CDs should be almost closely packed from one end to the other on the α,ωdimethacrylate PDMS chain. The mixture was stirred at room temperature for 24 h. The PPR complexes were obtained as white crystalline precipitate collected by filtration and then dried under vacuum (mass = 9.3912 g). The mass yield is 86%. 1H NMR in DMSO-d6, PDMS peaks: 0.0 ppm ((CH3)2Si−), 0.45−0.55 ppm (−CH2−Si), 1.6−1.7 ppm (−CH2−CH2−Si), 1.8−1.9 ppm (CH3− C), 4.0−4.1 ppm (−CH2−O), 5.5 and 6 ppm (CH2), and γ-CDs peaks: 3.5−3.6 ppm (m, H4), 3.6−3.7 ppm (m, H2), 3.8−3.85 ppm (m, H5), 3.85−3.95 ppm (m, H6), 3.9−4.0 ppm (m, H3), 4.3−4.5 ppm (t, OH6), 4.6−4.8 ppm (s, H1H anomeric), 5.3−5.6 ppm (2 × d, OH3 and OH2). Synthesis of the Densely Threaded Polyrotaxanes. γ-CD (3.48 g) and pyrene (0.543 g) were added in 10 mL of pure water and mixed at room temperature for 24 h. The solution became turbid due to the formation of a light yellow precipitate. The solution was named as the additional complex γ-CD/pyrene, i.e., polyrotaxane (PPR), and was then used for the synthesis of polyrotaxane (PR). A powder mixture of PDMS/γ-CD based PPR (1.69 g), 1-pyrenebutyric acid Nhydroxysuccimide ester (0.116 g), and sodium persulfate (0.411 mg) was prepared into a 100 mL round-bottom flask. Addition of sodium persulfate as an oxidant and a source of radicals allows also to grafted a controlled number of sulfate groups on CD, increasing the solubility of neo-formed macromolecular complex.28 The reaction started by adding 10 mL of the aqueous 1:1 pyrene/γ-CD complexes solution B

DOI: 10.1021/acs.macromol.6b00492 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules Scheme 1. Radical End-Coupling Synthesis Scheme of γ-CD/PDMS-Based Polyrotaxane Synthesized in This Study

MALDI-TOF MS. Characterization of PR by MALDI-TOF mass spectrometry was performed using a Applied Biosystems Voyager-DE Pro STR time-of-flight mass spectrometer (Applied Biosystems/MDS Sciex, Foster City, CA), equipped with a nitrogen UV laser (λ = 337 nm) operated in both the positive and negative ion reflector modes with an accelerating potential of ±20 kV and a grid percentage equal to 70%. Mass spectra were recorded with the laser intensity set just above the ionization threshold (2800 in arbitrary units, on our instrument) avoiding fragmentation and maximizing the resolution (pulse width 3 ns). Time delay between laser pulse and ion extraction was set to 450 ns. A set of parameters in the linear mode was also tested consisting of an accelerating potential of ±25 kV, a grid percentage of 93%, and an extraction delay of 800 ns, and the laser power was adjusted to 3200. Typically, mass spectra were obtained by accumulation of 200−1000 laser shots according to detection mode for each analysis and processed using Data Explorer 4.0 software (Applied Biosystems). Polyrotaxane stocks were prepared at 76 mg/mL in DMF and further diluted if necessary. Sinapinic acid (SA) was used as crystalline matrix and the stock prepared at 20 mg/mL in methanol/water 1/1 (v/v). THAP/TMG2 was prepared as previously described.28 Briefly, THAP was mixed with TMG at a molar ratio of 1:2 in methanol. The solution was then sonicated for 15 min at 40 °C. After removal of methanol by centrifugal evaporation in a SpeedVac for 3 h at room temperature, ILMs were left in a vacuum overnight. Final solutions were then prepared at a concentration of 90 mg/mL in DMF for use as a matrix. Samples for MALDI-MS analysis concentration were prepared by mixing 0.5−1 μL of sample and one volume 1/1 (v/v) of matrix. Then, 1 μL of the mixture was deposited via the “dried droplet” method on a mirror polished stainless steel MALDI target and allowed to dry at room temperature and atmospheric pressure for 5−20 min. External calibration was performed using proteins mixture provided by the manufacturer.

previously prepared and 5 mL of DMSO/Et2O (50:50 v/v). The mixture was stirred at room temperature for 18 h and then quenched by dipping the reaction flask into liquid nitrogen followed by a freezedrying step. 80 mg of pure PR was isolated by preparative SEC for characterizations by NMR and mass spectrometry. Optimized conditions were related to run 5 in Table 1. Characterization by SEC, NMR, and MALDI-TOF MS. Size Exclusion Chromatography. A first purification step was performed by size exclusion chromatography (SEC) analysis of PR in dimethylformamide (DMF) using a Styragel column (HR 4E, 5 μm, 4.6 × 300 mm; ref WAT045810) purchased from Waters (Saint-Quentin-enYvelines, France) coupled with UV (λ = 345 nm) and refractive index detection. The experiments were performed at a flow rate of 0.3 mL/ min, and the injection volume was 50 μL (1 mg/mL). Data extract from such SEC analysis led to the estimation of the conversion yield of the PR synthesis. This form of quantification is close to the quantification of PR conversion by direct titration of residues like free γ-CD after calibration. A second preparative purification of polyrotaxane was achieved by SEC in DMF using two serial preparative Ultrastyragel columns (103 Å, 5 μm, 19 × 300 mm; ref WAT025861) purchased from Waters (Saint-Quentin-en-Yvelines, France) at a flow rate of 2 mL/min and an injection volume of 400 μL (60 mg/mL). 1D 1H, 13C and 2D 1H−1H, and 1H−13C NMR. 1D 1H, 13C (DEPTQ-135), and 2D heteronuclear correlation 1H−13C HMBC, HSQC, and NMR experiments were performed at 400 MHz using a Bruker AVANCE III HD 400 spectrometer equipped with direct broadband (+19F) cryoprobe (with nitrogen-cooled RF coils). Measurements were performed at 298 K. 1H NMR data spectra were collected using 32K data points. Samples were dissolved in deuterated dimethyl sulfoxide (DMSO-d6) used also as an internal standard. Additional 1D 1H NMR, 2D 1H−1H NOESY, and 1D saturation transfer difference (STD) experiments were recorded in DMSO-d6 at 298 K on a Bruker Avance 600 MHz NMR spectrometer equipped with a cryoprobe. One-dimensional spectra were acquired with 64 scans and 16 000 data points. The NOESY experiments were carried out with a mixing time of 200 ms, 2048 data points × 256 increments × 128 scans, and a spectral width of 9 kHz in both dimensions. The data were zero-filled to give 4096 × 512 data matrix prior to FT conversion. For the 1D STD-NMR experiments, the PDMS chain resonances were saturated at 0.008 and 8.13 ppm (40 ppm for the reference spectra) with a cascade of 40 selective Gaussianshaped pulses of 50 ms duration with a 1 ms delay between each pulse, resulting in a total saturation time of 2.04 s. Subtraction of the saturated spectra from the reference spectra was performed by phase cycling. A STD control experiment has been performed with a saturation at −2 ppm (Figure 7A, green trace). Excitation sculpting sequence was used for suppression of the residual water signal at 3.6 ppm for all spectra.29

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RESULTS AND DISCUSSION Optimization of the Polyrotaxane Synthesis. Starting conditions were drawn from our previously reported study dealing with the radical blocking reaction of a fully α-CD packed PEO-based PR in high conversion yield (96%).27,30 This almost quantitative yield of PR formation was actually achieved by reaction of 1-pyrenebutyric acid N-hydroxysuccinimide ester (PBS) on the methacrylate telechelic functions of the PEO polymer chain previously threaded with α-CDs constituting a model reaction. As a primer, the aforementioned methodology was used with some minor modifications to synthesize PDMS-based PR using γ-CD as macrocycles. Unfortunately, a mere translation of the best operating conditions from PEO to PDMS did not lead to high C

DOI: 10.1021/acs.macromol.6b00492 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules yield PR synthesis (Figure S1, Supporting Information). This result was presumably assigned to the different solubility behavior between PDMS and PEO. Furthermore, it was anticipated that dethreading of the larger γ-CDs before the occurrence of the coupling reaction actually took place. Taking into account all these limitations, the synthesis route was optimized to increase the conversion yield from the pseudopolyrotaxane to polyrotaxane (Scheme 1). The coupling efficiency was estimated by SEC analysis of the crude reaction products, and main optimization steps are summarized in five distinct runs (Table 1). In the previously reported PEO-based PR model reaction, a DMSO/H2O 33/66 (v:v) mixture was efficient to achieve reaction. Such conditions were applied to PDMS-based PR synthesis (run 1, Table 1) but only led to very low conversion yield (2%). Unfortunately, the insolubility of PDMS in such mixture suggests using an additional cosolvent to improve the accessibility of the PDMS chain ends by the blocking groups. Moreover, the influence of various conditions was further examined (Table 1). Adding Et2O as cosolvent (DMSO/Et2O/H2O 16.5/16.5/66 v:v:v) (run 2, Table 1) considerably improved solubility of the PDMS. Nevertheless, an important dethreading of the γ-CDs still occurred and was detected by SEC analysis (Figure S1). Examination of the chromatograms suggested that only 14% (w/w) of the PPR was effectively transformed into PR. The conversion yield was improved by saturating the reaction medium with additional 180 mM γ-CDs (run 3, Table 1). We postulate that the equilibrium could shift toward the formation of the supramolecular complex and consequently limit the dethreading of the molecular macrocycles.31 However, it was observed that instead yield was increased to 27% (w/w) at those concentrations; the medium was quite heterogeneous, impairing the probability of coupling occurrence. To ensure a homogeneous medium, the reaction mixture was diluted 5-fold, while preserving the saturation in γ-CDs ([PPR]/[PBS] from 10/100 to 2/20, mol/mol) and adjusting the amount of Na2S2O8 keeping constant the initiator concentration (5% w/w). The saturation in γ-CDs and the reactant concentrations have been kept constant to avoid the dethreading which is favored in diluted medium as previously reported.27 Such simple adjustment led to sharply increased yield to 78% (w/w) (run 4, Table 1). In all runs, an excess of blocking agent (PBS) was used as compared to the number of PDMS extremities (nPDMS extremities = 5nPBS). However, it cannot be excluded that the blocking agents (pyrene derivative) could have been trapped into the hydrophobic cavities of the γCDs, present in large amount, thus limiting the reaction efficiency on chain ends (run 4, Table 1). This hypothesis was investigated in the last run, where the cavities of the γ-CDs present in large scale used to saturate the reaction medium were previously filled with pyrene molecules ([γ-CD]/[pyrene] from 180/0 to 180/180 mM/mM). In this case, the conversion yield slightly increased up to 81% (w/w) constituting the best results obtained for PR synthesis ends (run 5, Table 1). The low difference in PR conversion yield from run 4 to 5 can be explained by the large excess of PBS used at each experiment, leading to a good reactivity of the PBS despite the loss of PBS trapped by CDs. This fact is more obvious for run 4 than run 5, where the saturation of the medium by the complex γ-CD/ pyrene limits the PBS trapping by free CDs, leading to a number of CD borne by the polymer chain increasing from 13 to 22 (Table 2).

Table 2. Recapitulative of Polyrotaxanes Features Obtained by SEC Analysis run no.

Mwa (kDa)

Mnb (kDa)

PDIc

nCDsd

%FRe

1 2 3 4 5

480.0 78.0 80.5 21.4 52.7

368.7 52.4 59.4 23.2 35.6

1.29 1.49 1.35 1.60 1.47

NC 34 39 13 22

NC 83 95 32 54

a

M̅ w of the PPR was estimated to be equivalent to the full packed PPR.21,22 bM̅ n of the PPR was estimated to be equivalent to the full packed PPR.21,22 cThe polydispersity index (PDI) is obtained by M̅ w/ M̅ n. dnCDs is the estimated average number of γ-CDs threaded. e%FR is the estimated average threading degree. NC: not calculable.

Although the conversion yield was optimized, the crude product still contains a large amount of free CDs, pyrene molecules, and PBS derivatives as evidenced by SEC chromatograms (Figure 1).

Figure 1. SEC chromatograms of polyrotaxane from run 5 using two serial preparative columns analyzed in DMF as crude product (blue trace) and after a first extraction by SEC (black trace).

It must kept in mind that without blocking the PDMS extremities, the PDMS/γ-CD inclusion complex breaks in SEC experimental conditions. The PPR SEC chromatogram in DMF exhibits a unique peak corresponding to the free CDs since sensitivity for PDMS in DMF is too low to observe the polymer (Figure S1). The high molar mass peak observed in the different runs is attributed to the PR. Depending on the nature of the polymer backbone, molecular weight, polydispersity index, and the number of CDs threaded, the elimination of the free macrocycles still remained a quite challenging task. Various methods commonly used to purify polymers such as precipitation, filtration, ultrafiltration, dialysis, etc., were unsuccessful to eliminate free CDs. Such limitations were demonstrated by preparative SEC analysis of the crude samples, which always showed the presence of the residual free γ-CDs (Figure 1, blue trace). These results suggested the formation of strong aggregates between PR and free CDs, which persist even in the presence of hydrogen bonds disrupting agent such as lithium salts or urea (100 mM). After collecting fractions of the peak corresponding to PR, subsequent injections on preparative SEC successfully led to pure PR without free γ-CDs (Figure 1, black trace). All features obtained by SEC, concerning macromolecular edifices obtained by applying the five aforementioned runs, are listed in Table 2. Initial conditions, D

DOI: 10.1021/acs.macromol.6b00492 Macromolecules XXXX, XXX, XXX−XXX

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Figure 2. Positive MALDI-TOF mass spectrum obtained in linear mode (A) and in reflector mode (B) with pure polyrotaxane (3.8 mg/mL) from run 5 mixed with THAP/TMG2 both in DMF.

obtained verifying the presence of γ-CDs threaded on the PDMS polymer chain, but more importantly confirming the blocking reaction ensuring the efficient formation of the polyrotaxane structure. A characteristic MALDI-TOF mass spectrum acquired in linear mode is shown in Figure 2A. Detectable peaks were seen in the range of m/z 3052−26 400 with an average 1300−1500 mass units shift corresponding to the unmodified, mono- or disulfated γ-CD molecular weight. The difficulty to desorb high molecular weight polyrotaxane, especially with common solid matrices as sinapinic acid (SA) (Figure S2A), and the importance to screen various matrices in order to optimize the detection were previously investigated.28 Taking into account the difficulties to get reliable mass spectrum for high molecular weight compounds, identification of species has been carried out on the first distribution which corresponds to PR with one γ-CD. Furthermore, MS acquisitions were also carried out in reflector mode which offers better resolution and improved mass accuracies for the detection of compounds exhibiting molecular weight typically