Analytica Chimica Acta 808 (2014) 144–150

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Mass Spectrometry

Direct TLC/MALDI–MS coupling for modified polyamidoamine dendrimers analyses Emma-Dune Leriche a , Marie Hubert-Roux a,∗ , Martin C. Grossel b , Catherine M. Lange a , Carlos Afonso a , Corinne Loutelier-Bourhis a a Normandie Université, COBRA, UMR6014 and FR3038, Université de Rouen, INSA de Rouen, CNRS, IRCOF, 1 rue Tesnière, 76821 Mont-Saint-Aignan Cedex, France b University of Southampton, School of chemistry, Highfield, Hants, SO17 1BJ Southampton, UK

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• TLC separates the species present in polyamidoamine samples according to their polarity. • TLC/MALDI allows us to attribute more easily the fragment ions as they are aligned with their precursor ions with the same Rf. • TLC/MALDI is easy, fast and requires small quantities of material.

a r t i c l e

i n f o

Article history: Received 28 June 2013 Received in revised form 13 September 2013 Accepted 18 September 2013 Available online 4 October 2013 Keywords: Polyamidoamine dendrimers TLC MALDI Tandem mass spectrometry

a b s t r a c t Polyamidoamine (PAMAM) are synthetic dendrimers which present attractive properties for the biological and biomedical fields, as they proved to be efficient drug and gene carriers. In order to increase their transfection efficiency, chemical modifications of the amino end-groups had been reported. In this work, the synthesis of the ammonia-cored G1(N) PAMAM and the consecutive chemical modification with glycine or phenylalanine amino-acids were monitored using the coupling of thin layer chromatography (TLC) with matrix–assisted laser desorption ionization–mass spectrometry (MALDI–MS). Thus, the monitoring of the PAMAM synthesis included the identification of the by-products such as defective structures of PAMAM dendrimers as well as the study of phenylalanine-grafted PAMAM oligomer distribution. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Since its introduction on silica gel by Stahl, thin-layer chromatography (TLC) is an analytical method, which is still broadly used in various fields such as food analysis, pharmaceutical research, organic and clinical chemistry [1,2]. This technique presents the advantage to be fast, inexpensive and constitutes an easy-to-perform approach. As the solvent consumption is relatively

∗ Corresponding author. Tel.: +33 235522924. E-mail address: [email protected] (M. Hubert-Roux). 0003-2670/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2013.09.037

low, which could be lower than conventional HPLC, it could be considered as an environment-friendly analytical method. Another advantage compared to HPLC is that memory effects can be avoided as the stationary phase is renewed for each analysis [3]. Conversely, the main drawbacks are the low resolution of separation compared to HPLC and the poor reproducibility of the retardation factor (Rf). Consequently, the systematic use of standards is necessary for spot identification [3]. The coupling with mass spectrometry (MS) permits to overcome this limit because MS provides analyte identification and structural information. First indirect TLC/MS analyses were described in the 1980s. They involved numerous preparative steps, including TLC spot scraping

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Scheme 1. Structure of glycine or Boc-phenylalanine-modified PAMAM G1(N).

and extractions in suitable solvents, before analyses of the extracts using ionization methods such as electron ionization or chemical ionization [4]. In the early 1990s, the first direct TLC/MS couplings were performed using second ion mass spectrometry (SIMS), fast atom bombardment (FAB) or laser desorption (LD) [5]. The coupling of TLC with matrix-assisted laser desorption ionization (MALDI) was reported for the first time by Gusev et al. [4]. These authors developed various methods for applying the matrix on the TLC plate, either by depositing the matrix solution after analyte extracting onto the plate surface or by pressing a matrix layer onto the TLC plate previously crystallized in a stainless steel support [5,6]. More usually, the matrix solution can be sprayed on the TLC plate [7]. Most applications of this coupling concerned the analyses of biomolecules such as phospholipids [8,9], peptides [4–6,10], oligosaccharides [11] or drugs in biological samples [7,12]. Despite the simplicity and effectiveness of this technique, only few studies reported TLC/MALDI analyses for synthetic polymer characterization. TLC separation of polymers depends mostly on the structure of the end-groups rather than on the degree of polymerization [13]. Thus, the TLC/MALDI coupling permits to determine the end group functionalization of polybutadiene, polystyrene [13] as well as poly(methyl)methacrylate [14]. TLC and MALDI/MS were also combined to achieve characterization of synthetic polyether mixtures. Thus, end-groups and repeating units were determined for the main expected product as well as for characterization of impurities and by-products [15]. Polyamidoamines (PAMAM) are synthetic dendrimers composed of a core, repeating units and terminal functional groups [16]. These dendrimers have potentially many applications in biomedical fields as drug and acid nucleic carriers. However, these compounds present some cytotoxicity that limits their medical use. In order to decrease such residual toxicity and improve their transfection efficiency, chemical modifications of their end-groups have been reported [17,18]. The grafting of amino acid residues on their chain ends to form heterobifunctional dendrimers constitutes one of the possible chemical modifications [19]. Our group had developed chemical modification of first-generation (G1) PAMAM with various amino-acid residues. For this purpose, the ammonia- as well as ethylene diamine-cored PAMAM have been synthesized. Here, we present the TLC/MALDI coupling for monitoring the synthesis and the chemical modification of G1 PAMAM with glycine and phenylalanine residues. Glycine was chosen as a model molecule to optimize the chemical modification protocol while phenylalanine was selected for its aromatic side-chain, which could induce specific interactions with DNA structures [18]. TLC/MALDI coupling is also used to monitor sample quality of both the unmodified- and modified-PAMAM dendrimers. Recently, a LC/ESIQTOF-MS method has been developed to identify and quantify PAMAM dendrimers of generations 0 to 3 [20,21]. Compared to this robust and reliable method, TLC/MS presents the advantage to be fast, easy and requires low amount of raw materials. Furthermore,

MALDI spectra are less complex than ESI spectra since mainly singly charged ions are produced by MALDI ionization. Thus, TLC/MALDI could be a good alternative approach to monitor PAMAM synthesis. Thus, to fully investigate the potential of this technique, we analyzed three different samples of PAMAM dendrimers: the first-generation ammonia-cored PAMAM G1(N), the completely glycine modified first-generation ammonia-cored PAMAM Gly6 G1(N) and the N-(tert-Butoxycarbonyl) phenylalanine modified first-generation ammonia-cored PAMAM N-Boc Phen G1(N) (Scheme 1). To our knowledge, this work constitutes the first direct TLC/MALDI combination for dendrimer characterization. 2. Experimental 2.1. Polymer synthesis PAMAM G1(N) was prepared according to Tomalia synthesis [16]. Syntheses of Glycine modified G1(N) and Boc-phenylalanine modified G1(N) were adapted from Wang et al. [17]. All these syntheses will be described in a future paper. 2.2. Thin-layer chromatography HPTLC Silica gel 60 F254 plates (5 × 7.5 cm in size on aluminium backs) were purchased from Merck (Darmstadt, Germany). PAMAM dendrimer samples were dissolved in methanol at a concentration of 40–60 mg L−1 . Then, 0.9 ␮L of each sample was spotted onto the TLC plate. For PAMAM G1(N) and glycine modified PAMAM G1(N) (Gly6 G1(N)), TEA/H2 O/MeOH (10/45/45) were used to develop the plates. For the Boc-Phen PAMAM G1(N), H2 O/MeOH (10/90) was used as the developing solvent. Once the separation was achieved, the TLC plate was dipped vertically twice in a matrix solution of 2,5dihydroxybenzoic acid (200 mg mL−1 in acetonitrile/H2 O (90/10: v/v)). After drying, the TLC plate was inserted into the TLC target adapter (Bruker, Bremen, Germany). 2.3. Mass spectrometry MALDI–TOF(/TOF)–MS data were performed on an Autoflex III time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a frequency-tripled Nd:YAG laser emitting at 355 nm. FlexControl (v3.3) software including TLC/MALDI software tool was used for data acquisition. FlexAnalysis (v3.3) and SurveyViewer (v1.3) softwares were used for data reprocessing. SurveyViewer allowed to extract ion chromatograms from the full TLC/MALDI plate data. Spectra were acquired in the positive-ion reflectron mode at 100 Hz laser shot frequency. The accelerating voltage was set to 19 kV and the extraction delay time was set to 40 ns in MS mode. In this mode, automatic acquisitions were performed with 500 laser shots per raster position at 50% laser fluence and spectra recorded every mm (X-step = 1 mm). Thus, TLC plate

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Fig. 1. Picture of the MALDI/TLC adapter with the plates of (A) G1(N) and (B) Gly6 G1(N). The individual spots were drawn according to the results from TLC/MALDI–MS. The color code corresponds to the structures in Fig. 2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

was raster scanned with the laser in an automated manner to collect a data set. For fragmentions analysis in the tandem time-of-flight (TOF/TOF) mode, the recorded mass spectra were the result of 1000 spectra averaged in the parent mode at 50% laser fluence and 1000 spectra in the fragment mode at 65% of laser fluence. The precursor ion kinetic energy was 8 keV. The ion selection was carried out with a time ion gate. Fragment ions generated by Laser Induced Dissociation (LID) of the precursor were further accelerated by 19 kV in the LIFT cell allowing a full product ion spectrum to be recorded. External calibration of MALDI-TOF mass spectrometer was carried out using a mixture of peptides. 3. Results and discussion 3.1. Optimization of TLC conditions The first TLC experiments were performed with different organic solvents such as THF, acetonitrile or methanol as eluents. However, they were not sufficiently polar to allow the migration of the PAMAM dendrimers. Thus, the addition of 10% of water in methanol was required to elute the less polar N-Boc phenylalanine G1(N). For the most polar compounds, G1(N) PAMAM and glycine modified G1(N) PAMAM, a larger proportion of water in methanol was necessary as well as the addition of triethylamine (TEA) within the ratio (45/45/10: H2 O/MeOH/TEA). The use of TEA, (pKa 10.65), permitted to increase the basicity of the solvent system and consequently to deprotonate the silanol groups of the TLC silica gel. Thus, ionic adsorption of the TEA amino groups onto the anionic silica gel could occur yielding “TEA/anionic stationary phase ion” pairing formation [22]. The resulting neutral surface modified stationary phase permitted to prevent the detrimental ionic adsorption of the basic PAMAM dendrimers (pKa ≈ 9.2 [23]) since adsorption could hinder the migration and separation of PAMAM.

of the analyte over migration distance of the solvent front) values were, respectively, of 0.2 and 0.6 indicating that, at least, two compounds were expected. The mass spectrum extracted from the first spot, corresponding to the most polar compound(s) (Rf value = 0.2), showed ions at m/z 1045.0 and 1067.0 which, respectively, corresponded to the protonated molecule ([M + H]+ ) and the sodium adduct ([M + Na]+ ) of the ideally branched structure of G1(N) (Fig. 2a). It constituted the major species according to the abundance of the extracted ion chromatograms of m/z 1045.0 and 1067.0. The first spot (Rf value ≤ 0.2) also showed a minor species, which corresponded to a dimeric defective structure [Dimer-60] which was not separated from G1(N) because of their similar polarity. The [M + Na]+ ion of this dimer was detected at m/z 2050 (data not shown). The second spot (Rf value = 0.6) displayed ions at m/z 984.9 and 1006.9, which corresponded to the protonated molecule and the sodium adduct of the well-described defective structure [G1(N)-60] (Fig. 2b), previously evidenced by off-line capillary electrophoresis MALDI/MS coupling [24]. This defect noted D’1 was formed by intramolecular cyclization with an ethylene diamine molecule (EDA) missing (60 Da) (Scheme 2). The LID spectra of both m/z 984.9 ([D 1 + H]+ ) and 1006.9 ([D 1 + Na]+ ) precursor ions (data not shown) were identical to those previously obtained by CZE/MALDI–MS2 showing the neutral loss of defective loop (270 Da) which is characteristic of this defective structure [24]. Another compound, which [M + H]+ and [M + Na]+ ions were detected at m/z 528.4 and 550.4, belonged to the second spot and was eluted at a Rf value slightly higher than that of the [G1(N)-60] defect. This compound could be identified as the defective structure of G1(N) that resulted from the intramolecular cyclization involving losses of one EDA molecule, one branch (−114 Da) and one arm (−342 Da) [G1(N)-114-342-60] (Fig. 2c, Scheme 2). It constituted one of the most common PAMAM defective structures exhibiting a defective loop formed from the reaction between one EDA molecule and two end branches of a half-generation [25,26]. The most apolar compound that could be evidenced in our TLC/MALDI conditions had a Rf value of 0.74 and was also eluted within the second spot. The corresponding [M + Na]+ ion was detected at m/z 946.9. This compound could correspond to the defect of G1(N) formed after the losses of two EDA molecules [G1(N)-2 × 60] (Fig. 2d, Scheme 2). All the detected defective structures have been previously described for commercial PAMAM samples [25,26]. Note that the Rf values of the defective and ideally branched structures are in agreement with their polarity, which increases with the number of amino groups present in the surface. Actually, the [Dimer-60] defect which is the most polar compound contained 10 amino groups and migrated at the lowest Rf value while the most apolar compound, [G1(N)-2 × 60] defect which held only two amino groups, migrated at the highest Rf value. In comparison with CZE/MALDI, the TLC/MALDI approach permitted to detect a higher number of components; the presence of three defects in addition to the ideally branched PAMAM and the previously mentioned defect could be established [24]. Moreover, all the species could be identified by MS/MS experiments. Contrary to the CZE collected fractions which contained phosphate buffer required for separation, no salt or buffer were required for TLC. In consequence, the sensitivity was higher in TLC/MALDI than in CZE/MALDI.

3.2. TLC/MALDI analyses 3.2.1. G1 ammonia-cored PAMAM [G1(N)] TLC/MALDI experiments were performed by raster scanning the TLC plate with the laser in an automated manner. TLC/MALDI analysis of the synthetized PAMAM G1(N) (Fig. 1A) revealed the presence of two main spots which retardation factor (Rf: migration distance

3.2.2. TLC/MALDI analysis of glycine modified G1-ammonia-cored PAMAM (Gly6G1(N)) The same solvent system containing TEA was used for the fully modified Gly6 G1(N) PAMAM. Under these conditions, TLC/MALDI analysis showed the presence of two main spots which Rf values were 0.3 and 0.48 (Fig. 1B). The mass spectrum of the most

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Fig. 2. Positive ion MALDI–TOF mass spectra recorded directly on the TLC plate for PAMAM G1(N): (a) ideal unmodified G1(N) and three defect structures (b) D 1 , (c) D 2 and (d) D 3 .

polar compound (Rf value of 0.3) showed the ions at m/z 1387.2 and 1409.2 which, respectively, corresponded to the [M + H]+ and [M + Na]+ ions of completely modified Gly6 G1(N) PAMAM (Fig. 3a). Another low-abundant ion was observed at m/z 1238.1 and was 171 m/z units down-shifted relative to m/z 1409.2. This ion could rather arise from in-source decay (ISD) fragmentation of m/z 1409.2 (neutral loss of glycine-modified branch) than from glycine grafting onto one of the previously mentioned defects because none of these structural defects could yield a modified by-product exhibiting [M + H]+ or [M + Na]+ at m/z 1387.2. Moreover, the MALDI–MS2

spectrum of the m/z 1409.2 precursor ion (Fig. 4a) displayed the corresponding product ion at m/z 1238.1 as well as other product ions explained by the characteristic PAMAM fragmentation pattern involving successive losses of either modified-branch or modifiedarm (Scheme 3). Thus, m/z 1409.2 dissociated with neutral loss of modified-branch yielding the m/z 1238.2 product ion which could subsequently dissociate into m/z 952.8 by elimination of modifiedtwo branches moiety. The m/z 952.8 could also directly arise from the sodium adduct at m/z 1409.2 by neutral loss of fully modifiedarm. Then, m/z 952.8 product ion could yield m/z 508.3 and 223.1 by

Scheme 2. Structures of ideally branched and defects of G1(N).

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Fig. 3. Positive ion MALDI–TOF mass spectra recorded directly on the TLC plate for the completely glycine modified PAMAM G1(N) [Gly6 G1(N)]: (a) Ideal structure of glycine modified PAMAM G1(N) and (b) the main defective structure of glycine modified PAMAM G1(N), D  1 .

successive losses of fully modified-arm and modified-two branches moiety. The m/z 479.3 and 194.1 product ions, respectively, corresponded to the sodiated fully modified-arm and to the sodiated modified-two branches moiety. All these fragmentations were previously described by ESI–MSn analyses [27]. The other compound which migrated at a Rf value of 0.48 was less polar and gave ions at m/z 1213.1 and 1235.1 which could be, respectively, assigned to the [M + H]+ and [M + Na]+ ions of

the modified defective species D 1. Actually, this defect D 1 could hold four glycine residues after the chemical modification step and lead to the D 1 species (Fig. 3b). The MALDI–MS2 spectrum of m/z 1235.1 selected as precursor ion showed the successive neutral losses of modified-branch, modified-two branches moiety and fully modified-arm as well as the neutral loss of 270 Da corresponding to the elimination of the loop which is characteristic of the D 1 defective structure (Fig. 4b).

Fig. 4. LID spectra recorded directly on the TLC plate for the completely glycine modified PAMAM G1(N) [Gly6 G1(N)]: (a) ideal structure of glycine modified PAMAM G1(N) [Gly6 G1(N)] m/z 1409.2 and (b) main defect of glycine modified PAMAM G1(N), D  1 m/z 1235.1; *metastable ions.

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Scheme 3. (a) Ideal structure of completely glycine modified PAMAM G1(N) [Gly6 G1(N)] (b) structure of the main defect of glycine modified PAMAM G1(N), D  1 . Product ions from sodium adducts are reported.

It could be noted that some mass inaccuracies for these experiments were observed and resulted to surface irregularities of the TLC plate, as previously reported in other papers [3].

(iii) Boc-Phe2 G1(N) with Rf value of 0.66 ([M + H]+ and [M + Na]+ ions detected at m/z 1539.0 and 1561.0) (Fig. 5c), (iv) Boc-Phe3 G1(N) with Rf value of 0.85 ([M + Na]+ ion detected at m/z 1808.9) (Fig. 5d).

3.2.3. TLC/MALDI analysis of the Boc-phenylalanine modified first-generation (G1) ammonia-cored PAMAM (BocPhen-G1(N)) First assays showed that the deprotected sample, Phen -G1(N), was too polar to have an effective separation (data not shown). Thus to monitor the chemical modification, in particular to study the distribution of grafted phenylalanine residues, the TLC was performed by spotting the BocPhen -G1(N) sample. The TLC/MALDI analysis (Fig. 5) revealed the presence of four spots which were identified as:

To demonstrate the benefit of TLC/MALDI coupling, conventional MALDI–TOF/MS experiments were performed using the dried droplet deposit on the MALDI stainless steel target plate for BocPhen -G1(N) sample. The mass spectra showed sodiated adducts of the oligomer distribution of modified PAMAM BocPhen -G1(N) with n = 0 to 2 (Fig. 6). However, it is to notice that the BocPhe3 G1(N) was very randomly detected, depending on the position of the laser shot on the MALDI target. This phenomenon is wellknown for MALDI ionization because the analyte signal produced in MALDI depends strongly on the analyte and on the sample preparation [28–30]. However, the stainless steel MALDI target and TLC plate have very different surfaces which can lead to different ionization efficiency. Thus, the coupling with TLC seemed to allow to reduce these discrimination effects and to ensure detection of all species present in the sample. More importantly, many ions present

(i) unmodified G1(N) with Rf value of 0.2 ([M + H]+ and [M + Na]+ ions detected at m/z 1044.8 and 1066.7, respectively) (Fig. 5a); (ii) Boc-Phe1 G1(N) with Rf value of 0.41 ([M + H]+ and [M + Na]+ ions detected at m/z 1291.8 and 1313.9, respectively) (Fig. 5b);

Fig. 5. Positive ion MALDI–TOF spectra recorded directly on the TLC plate for the N-Boc Phenylalanine PAMAM G1(N): (a) unmodified G1(N) (b) BocPhe1 G1(N) (c) BocPhe2 G1(N) d) BocPhe3 G1(N); *metastable ions.

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Fig. 6. Positive ion MALDI–TOF spectrum of the N-Boc Phenylalanine PAMAM G1(N); *metastable ions.

in the MALDI spectrum were difficult to attribute, as they do not correspond to expected dendrimers or defective structures. After TLC separation it is obvious that these ions were produced by insource decay. In this case each fragment ions is associated with their precursor ion aligned within the same Rf thanks to the TLC separation. The coupling is even more adequate since DHB is known to be a matrix producing highly in-source decay fragments [31]. One can notice that, in the case of phenylalanine, the grafting yielded an oligomer distribution of Boc-Phen G1(N), the hugest species corresponding to the grafting of three phenylalanine residues, contrary to glycine grafting which gave the completelymodified Gly6 G1(N) PAMAM. That could be explained by higher stearic hindrance encountered for Boc-Phen G1(N) formation. 4. Conclusion Direct TLC/MALDI coupling was used for the first time to characterize PAMAM dendrimers. This analytical tool is particularly powerful to monitor the synthesis and chemical modification of PAMAM. It was specifically efficient to show the presence of dendrimer defective structures, inherent in the Tomalia divergent approach. Without the TLC separation, some of the higher mass dendrimers were not detected in the MALDI-TOF spectra and fragment ions produced by in-source decay can be difficult to attribute as they may have different origins. The TLC/MALDI coupling allows to increase the detection of these high mass dendrimers and to attribute more easily the fragment ions as they are aligned with their precursor ions with the same Rf. TLC/MALDI is easy, fast and usually requires small quantities of material. The automatic acquisitions every millimeter of the TLC plate allow to avoid another spot detection (UV, specific chemical derivatization. . .) before the MALDI analysis. Acknowledgments The authors thank the Region Haute-Normandie, the Labex SynOrg (ANR-11-LABX-0029) and the European Regional Development Fund (ERDF 31708), IS:CE-Chem project and Interreg IV A France(Channel)-England for financial support.

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