In-Source Decay of HyperbranchedPolyesteramides in Matrix-Assisted LaserDesorption/Ionization Time-of-Flight MassSpectrometry

Dirk Muscat, Huub Henderickx, Gerard Kwakkenbos,Rolf van Benthem, and Chris G. de KosterDSM Research, Geleen, The Netherlands

Roel Fokkens and Nico M. M. NibberingInstitute of Mass Spectrometry, University of Amsterdam, Amsterdam, The Netherlands

Hyperbranched polyesteramides (DA2), prepared from hexahydrophthalic anhydride (D) anddiisopropanolamine (A) have been characterized, by use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), field desorption (FD)-MS, andelectrospray ionization (ESI)-MS. MALDI of polyesteramides produces protonated molecules.The spectra show a complex chemical composition distribution and end-group distributionwhich are mainly composed of two series of homologous oligomers DnAn11 2 mH2O andDnAn 2 mH2O, where m 5 1–2. Signals from protonated molecules DnAn11 and DnAn arealmost absent in the MALDI spectrum, whereas these ions are responsible for the base peak ofDnAn11 2 mH2O and DnAn 2 mH2O (m 5 1–2) clusters in the ESI spectrum. The absence of–OH end-groups signals in the MALDI spectrum is due to a metastable decay of protonatedDnAn11 and DnAn ions in the ion source of the MALDI mass spectrometer prior to ionextraction. In-source decay results in the formation of protonated lower DnAn11 2 mH2O andDnAn 2 mH2O oligomers and their corresponding neutrals, leading to wrong conclusionsconcerning the relative end-group distribution as a function of the degree of polymerizationand the chemical composition. (J Am Soc Mass Spectrom 2000, 11, 218–227) © 2000American Society for Mass Spectrometry

Since the introduction of matrix-assisted laser de-sorption/ionization time-of-flight mass spectrom-etry (MALDI-TOF-MS) for the analysis of macro-

molecules [1, 2, 3], this particular mass spectrometrictechnique has been developed rapidly for structuraland compositional analysis of synthetic polymers. Thestrength of MALDI-TOF-MS analysis is the capability ofdirect oligomer resolved analysis of molecular weightdistributions (MWD). Improvements in resolution andaccuracy of mass determination by applying time-lagfocusing, also called delayed extraction, or coupling ofMALDI with Fourier-transform ion cyclotron resonancemass spectrometry (ICR) afford the qualitative determi-nation with oligomer resolved resolution [4] of chemicalcomposition including end-group identity [5] and end-group composition [6], the extent of cyclization thatoccurs at each degree of polymerization [7] and blocklength distribution [8, 9]. Improvements in matrix andsample preparation strategies have extended the mass

range for analysis of synthetic polymers by MALDI upto 1.5 million Da [10].

A conditio sine qua non for chemical compositionanalysis by MALDI is that the pseudomolecular ions ofthe oligomers do not dissociate by elimination of end-groups and/or lower oligomers. Although MALDI wasinitially described as a “soft ionization” process, asignificant extent of metastable fragmentation may oc-cur for MALDI-generated ions. Particularly, this frag-mentation occurs for peptide ions and is exploited toobtain sequence information for peptides. Spectra fromthe product ions of metastable decay in the first field-free region of a reflectron TOF-MS, also called post-source decay (PSD), are acquired by successively chang-ing the reflectron potential in discrete steps to correctfor the lower kinetic energy of the PSD ions. In this way,metastable ions within a specific mass range can bebrought in time focus at the detector. These massspectra are then combined into a single composite massspectrum. With precursor ion selection prior to thefield-free region via pulsed deflection methods, tandemmass spectrometry (MS/MS) of collisionally activated

Address reprint requests to Dr. C. G. de Koster, DSM Research, PO Box 18,6160 MD Geleen, The Netherlands. E-mail: chris.koster-de@dsm-group.com

© 2000 American Society for Mass Spectrometry. Published by Elsevier Science Inc. Received July 30, 19991044-0305/00/$20.00 Revised October 28, 1999PII S1044-0305(99)00141-5 Accepted October 29, 1999

(CA) ions can be performed. Significant amounts ofstructural information can be obtained for moderatemass-to-charge peptide ions via this technique.MALDI/PSD of mass selected ions with collision gashas been shown to be a practical method for thecharacterization of polymers and the determination ofthe end groups of poly(methyl methacrylate), poly(eth-ylene glycol), and poly(ethylene terephtalate) [11].

Post-source decay is not the only source of fragmentions that are generated in MALDI. Metastable decay inthe ion source prior to ion extraction is also an operativereaction mode upon MALD ionization. A considerableextent of metastable decay of large peptide ions andneutrals occurs within 300 ns after laser irradiation inthe ion source prior to ion extraction into the flight tube[12]. It is generally referred to as in-source decay (ISD).The ISD spectra of peptides contain a wealth of se-quence information [13, 14]. Factors influencing fastfragmentation were thoroughly studied by Brown et al.[15], and it was concluded that the fragment ionsobserved are not due to prompt fragmentation [16], butare a result of metastable decay on a fast time scale.Reflectron TOF MALDI spectra of several carbohy-drates show various well-focused fragment ions, partic-ularly when recorded with instruments fitted withtime-lag focusing [17, 18]. These ions have been shownto be the result of in-source fragmentation. Anothersource of fragment ions is laser induced pyrolysis of theanalyte. The pyrolysates produced can then be de-sorbed to form ions that are observed in the MALDIspectrum. In this report we will provide evidence thatin-source decay is not restricted to protonated peptidesand sodiated carbohydrates, but also occurs uponMALDI ionization of a particular class of syntheticpolymers, i.e., hyperbranched polyesteramides (HPEA).

Chemical Composition of Hyperbranched PolyesterAmides

Recently we reported the synthesis of HPEA [19] fromreadily available monomers like hexahydrophthalic an-hydride, phthalic anhydride, glutaric anhydride, etc.and diisopropanolamine. The synthesis of HPEA fromthe above mentioned monomers cannot be reduced to asimple AB2 (see below) case, although the first reactionstep, namely the addition of anhydrides to diisopro-panolamine might imply this. The first reaction stepleads to a series of multifunctional monomers andoligomers, as determined by gel permeation chroma-tography (GPC). The functional groups are mainlyhydroxyl and carboxylic acid, but also amine, through adynamic equilibrium already at room temperature,similar to d-hydroxyalkylamides [20, 21]. Thus, themost simple and dominant monomer in the reactionmixture after the first reaction step, is at best describedby an AB2 system, where A represents the acid groupand B the hydroxyl, beside some ABB9 monomers,where B9 refers to the amine.

Scheme 1 shows as an example the main additionproducts of hexahydrophthalic anhydride and diiso-propanolamine. The other polycondensation occurs viaoxazolinium ions (Scheme 2). The molecular weightcontrol is ensured by an excess of diisopropanolamineas a “chain stopper.” In order to obtain a definedstructure and to adjust the molecular weight by theexcess of diisopropanolamine, the equilibrium shownin Scheme 1 has to be on the left side, as the amine canalso react with the oxazolinium ion (side reaction,pathway B) instead of an acid group (desired reaction,pathway A).

The amount of chain stopper is decreased throughthe side reaction (pathway B), leading finally to gelationif occurring at too high an extent. MALDI-TOF massspectrometry is applied to characterize the chemicalcomposition of the HPEA reaction product. If only thedesired reaction via pathway A occurs, exclusivelysignals from molecules with a ratio of hexahydroph-thalic anhydride and diisopropanolamine n:n (cycles)and n:n 1 1 should be observed. Signals with ratios ofhexahydrophthalic anhydride to diisopropanolamine ofn:n 1 2, n:n 1 3, etc. indicate the side reaction. Inorder to monitor and control the reaction proceedings,MALDI was applied to characterize the reaction prod-ucts during which we encountered several difficulties.In this paper we discuss the results of the characteriza-tion and also the pitfalls of MALDI characterization ofthe HPEA’s.

Experimental

MALDI-TOF mass spectrometry was carried out usinga Perkin Elmer/Perseptive Biosystems Voyager-DE-RPMALDI-TOF mass spectrometer (PerSeptive Biosys-tems, Framingham, MA) equipped with delayed extrac-tion [22]. A 337 nm UV nitrogen laser producing 3 nspulses was used and the mass spectra were obtained inthe linear and reflectron modes. Samples were preparedby mixing 10 mL of tetrahydrofuran (THF) solution ofthe HPEA sample (2 mg/mL) with 30 mL of a solutionof 3 mg/L 2,5-dihydroxybenzoic (DHAB) acid in THF.1 mL of that solution was loaded on the gold sampleplate. The solvent was removed in warm air. PSDspectra were obtained by adjusting the voltage appliedto the reflector (mirror ratio setting). MALDI/PSD

Scheme 1

219J Am Soc Mass Spectrom 2000, 11, 218–227 IN-SOURCE DECAY OF HYPERBRANCHED POLYMERS

spectra record the fragment ions of spontaneously de-composing [M 1 H]1 ions, while MALDI/PSD/CA(collisional activation) mass spectra record the fragmentions of [M 1 H]1 ions following collision with argon asthe target gas. For obtaining the latter spectra, the argonpressure was such that the intensity of the selected ionpeak was reduced by 60%.

Field desorption (FD) mass spectrometry was carriedout using a JEOL JMS SX102/102A four-sector massspectrometer (JEOL, Akishima Tokyo, Japan), coupledto a JEOL MS/MP7000 data system. 10 mm tungstenwire FD emitters containing carbon microneedles withan average length of 30 mm were used. The sampleswere dissolved in THF and then loaded onto the emitterwith the dipping technique. An emitter current of 0–25mA was used to desorb the sample. The ion sourcetemperature was generally 90 °C.

Positive ion electrospray (ESI) mass spectra of hyper-branched polyesteramides were recorded using a PESciex API 150 single quadrupole mass spectrometer(Perkin Elmer Sciex, Toronto, Ontario, Canada). Thesamples were introduced through the electrospray in-terface by infusion of a methanol/water (75/25) solu-tion containing 0.1% acetic acid at 5 mL min21 with asyringe pump (Harvard Apparatus, Saint Laurent, Que-bec, Canada) nitrogen was used as nebulizing andcurtain gas. The capillary tip was maintained at 5 kV,while the orifice was set at 30 V. Mass spectra werecollected in full scan mode, scanning from m/z 100–2000in 30 s. The mass spectral data were processed with theMultiView 1.4 software (PE Sciex).

The synthesis of the hyperbranched polyesteramides is described in [19].

Results and Discussion

Polymerization of the AB2/ABB9 system allowing sidereactions as depicted in Scheme 2 leads to the formationof DnAm reaction products, where D is the diacid and Ais the diisopropanolamine. Combination of m and nwithout reaction conditional and mechanistic con-straints leads to a set of molecular masses for thereaction products as given in Table 1 for n 5 1 to 6 andm 5 1 to 6. The molecular masses are calculated by

M~DnAm! 5 n*~MD 2 18! 1 m*~MA 2 18! 1 Mend

(1)

where MD 5 172 Da is the molecular mass of hexahy-drophthalic acid, MA 5 133 Da is the molecular mass ofdiisopropanol amine and Mend 5 18 Da is the mass of awater end-group. Generally, synthetic polymers areprotonated or metal ion cationized upon MALD ioniza-tion. We varied the MALDI conditions by addingdifferent alkali and metal salts. Addition of Ag1 ions tothe MALDI matrix does not result in the formation ofsilver cationized hyperbranched polyesteramide mole-cules. Isotope patterns which are characteristic for silvercationized [M 1 Ag]1 pseudomolecular ions are notobserved in the spectra (data not shown). A typicalMALDI-TOF-MS spectrum without addition of a metalsalt and obtained in the reflectron mode is given inFigure 1.

Several series of structurally related homologousoligomers can be discerned on the basis of the MALDI-TOF-MS spectrum (Figure 1). The strategy used for theidentification of the protonated/cationized oligomers

Scheme 2

220 MUSCAT ET AL. J Am Soc Mass Spectrom 2000, 11, 218–227

and for determination of the chemical composition ofthe individual oligomers by extrapolation to zero oli-gomers has been discussed elsewhere [23, 24]. Thisstrategy has been applied in this study without anymodification. A series of pseudomolecular ions with amass increment of 269 pointing to a repeating unit of[DA 2 H2O] is observed at m/z 385, 654, 923, etc.Extrapolation to zero monomers gives a residual massof 116. This residual mass is the summation of theend-group mass and the mass of the ionizing species.The end-group and cation for this series of homologousoligomers are diisopropanolamine minus one watermolecule (115 Da) and a proton, respectively. Sodiumadducts at m/z 407, 676, 945, etc are not observed(Figure 1) and sodium cationization reactions appar-ently do not occur upon MALD ionization of hyper-branched polyesteramides built up from D and Amonomers. Addition of sodium iodide to the DHBmatrix did not induce the production of [M 1 Na]1

pseudomolecular ions. MALD ionization of hyper-branched polyesteramides produces protonated mole-cules [M 1 H]1. The m/z 385, 654, 923, etc series of ionsis assigned as protonated DnAn11. Two DnAn11 relatedseries are found at Dm/z 5 18 and Dm/z 5 36 lowerthan the [DnAn11 1 H]1 ions. These ions originatefrom structurally homologous DnAn11 which have ap-parently lost one or two water molecules somewhere inthe polymerization reaction. Assignments of the otherpeaks in the MALDI spectrum are presented in Table 2.

In summary, the MALDI spectrum shows a complexchemical composition and end-group distribution.These distributions are mainly composed of two seriesof homologous oligomers DnAn11 2 mH2O andDnAn 2 mH2O, where m 5 1–2. These results provideevidence for the conclusion that only the desired path-way A occurs in the synthesis of HPEA (Scheme 2).

Protonated DnAn11 and DnAn oligomers are almostabsent in the MALDI derived HPEA chemical compo-sition distribution. Apparently, substantial unexpectedend-group loss (H2O) occurs in the organic synthesis ofthe polyesteramides. The DnAn11 may lose water mol-ecules leading to the formation of allylic end-groups.For the DnAn a second mechanism may be operativewhich leads to the formation of DnAn 2 H2O ions. TheDnAn oligomers possess an equal ratio of D and Amonomers and as a result a free carboxylic moiety.Therefore, DnAn oligomers are capable of losing a watermolecule in an intramolecular cyclization reaction byester formation with a terminal OH group from an Amonomer. Note, that cyclization is forbidden as areaction mode for the DnAn11 oligomers. Intramolecu-lar cyclization reactions are well recognized in polyestercondensation reactions [25] and in hyperbranched poly-mer synthesis [26]. In contrast to a polycondensate witha linear architecture where cyclization gives termina-tion of growth by removal of the two active chain ends,a hyperbranched structure bears a large number ofremaining reactive chain ends that are available forfurther polymer growth. Consequently, cyclic speciesare found for every degree of polymerization.

The extensive loss of end-groups from DnAn11 andDnAn oligomers is not in agreement with NMR andend-group titration data (data not shown). A rationalefor this unexpected end-group loss is MALDI inducedfragmentation on a short time scale in the ion source ofthe time-of-flight mass spectrometer before the ions areaccelerated and extracted into the flight tube. It hasbeen observed that a considerable amount of metastabledecay of large peptide ions occurs within 300 ns afterlaser irradiation in the ion source prior to ion extractioninto the flight tube [27]. The polyesteramides may beconsidered as branched peptides. Therefore, rapid frag-mentation of protonated higher oligomers to loweroligomers is a plausible assumption for the observedend-group losses.

Evidence for rapid metastable fragmentation in theion source, also called ISD, was obtained by comparison

Table 1. Molecular masses of HPEA oligomers as a function of the number of hexahydrophthalic acid (D) monomers anddiisopropanol amine (A) monomers

D\A 1 2 3 4 5 6

1 287 402 517 632 747 8622 441 556 671 786 901 10163 595 710 825 940 1055 11704 749 864 979 1094 1209 13245 903 1018 1133 1248 1363 14786 1057 1172 1287 1402 1517 1632

Figure 1. MALDI-TOF mass spectrum of a hyperbranched poly-ester amide using a 2,5-dihydroxybenzoic acid matrix: D denotesa hexahydrophthalic acid monomer and A indicates a diisopropa-nol amine.

221J Am Soc Mass Spectrom 2000, 11, 218–227 IN-SOURCE DECAY OF HYPERBRANCHED POLYMERS

of the MALDI mass spectra with FD mass spectra andESI mass spectra. Furthermore, to obtain additionalevidence PSD experiments of mass selected oligomerswith and without the presence of collision gas werecarried out.

FD generates protonated oligomers. Peak assign-ments and relative peak intensities are given in Table 2.ESI also produces protonated oligomers. The ESI-MSspectrum is given in Figure 2. Assignments of the peaksand their relative intensities normalized to that of thebase peak are presented in Table 2. We will limit thediscussion to two cases: the D3A3 and the D3A4 oli-gomers. The relative end-group compositions calcu-lated from the MALDI, FD, and ESI mass spectra ofD3A3, D3A3 2 H2O, and D3A3 2 2H2O and the analo-gous D3A4 composition are given in Table 3.

The m/z 941 [D3A4 1 H]1 ion is the base peak of the[D3A4 2 mH2O 1 H]1 cluster in the ESI spectrum (Fig-ure 3, Table 3) and m/z 941 carries 76% of the ion currentof the D3A4 end-group distribution, whereas m/z 923

[D3A4 2 H2O 1 H]1 comprises 72% of the end-groupdistribution in MALDI. The apparent end-group loss inthe synthesis of the polyesteramides is less dramatic asit was initially revealed by MALDI. The FD determinedend-group composition is somewhere between theMALDI and ESI one. The maximum of the ion currentdistribution gradually shifts from lower m/z 923 ions inMALDI to m/z 941 ions in ESI. Formation of m/z 905[D3A4 2 2H2O 1 H]1 indicative of double water lossesfrom the end-groups in the course of the synthesis isnegligible. An assumption in this intercomparison ofend-group distributions is that the ionization and de-tection efficiencies within a DnAn11 cluster are equal.We do not expect that an allylic structural entitychanges the ionization efficiency within a DnAn11 clus-ter dramatically, i.e., that formation of gas-phase pro-tonated DnAn11 2 H2O would be favored with respectto protonated DnAn11 molecules. An important param-eter for the efficiency of ion formation in a gas phaseion–molecule reaction is the proton affinity of the site of

Table 2. MALDI-TOF-MS spectrum in the reflectron mode, FD mass spectrum, and ESI mass spectrum of a hyperbranchedpolyesteramide. The relative intensities are normalized with respect to that of the base peak

m/z Ion MALDI FD ESI

252 [DA 2 2H2O 1 H]1 317 143 13270 [DA 2 H2O 1 H]1 328 242 118288 [DA 1 H]1 — 150 39367 [DA2 2 2H2O 1 H]1 513 293 109385 [DA2 2 H2O 1 H]1 1000 313 140403 [DA2 1 H]1 299 405 1000500 [DA3 2 H2O 1 H]1 69 20518 [DA3 1 H]1 121 76521 [D2A2 2 2H2O 1 H]1 415 220 10539 [D2A2 2 H2O 1 H]1 276 297 92557 [D2A2 1 H]1 — 242 17636 [D2A3 2 2H2O 1 H]1 152 76 7654 [D2A3 2 H2O 1 H]1 660 542 88672 [D2A3 1 H]1 94 1000 410769 [D2A4 2 H2O 1 H]1 160 52787 [D2A4 1 H]1 315 197790 [D3A3 2 2H2O 1 H]1 122 345 28808 [D3A3 2 H2O 1 H]1 70 350 122826 [D3A3 1 H]1 — 84 26905 [D3A4 2 2H2O 1 H]1 49 337 27923 [D3A4 2 H2O 1 H]1 216 676 166941 [D3A4 1 H]1 33 784 5871038 [D3A5 2 H2O 1 H]1 — 174 431056 [D3A5 1 H]1 207 1291059 [D4A4 2 2H2O 1 H]1 34 308 321077 [D4A4 2 H2O 1 H]1 43 205 801095 [D4A4 1 H]1 26 — 171174 [D4A5 2 2H2O 1 H]1 15 201 201192 [D4A5 2 H2O 1 H]1 74 400 621210 [D4A5 1 H]1 18 278 2361307 [D4A6 2 H2O 1 H]1 — 95 321310 [D5A5 2 3H2O 1 H]1 — 91 91325 [D4A6 1 H]1 — 75 731328 [D5A5 2 2H2O 1 H]1 — 102 381346 [D5A5 2 H2O 1 H]1 — 67 611443 [D5A6 2 2H2O 1 H]1 — 109 231461 [D5A6 2 H2O 1 H]1 — 107 501479 [D5A6 1 H]1 — 79 100

222 MUSCAT ET AL. J Am Soc Mass Spectrom 2000, 11, 218–227

protonation. Proton affinities of the hyperbranchedpolyesteramides are not known. Comparison of theproton affinity (PA) of the amino acid leucine (PA 5217 kcal/mol [28]) with 2-butene (PA 5 182 kcal/mol[29]) shows that the amide bond is the thermodynami-cally favored protonation site. Moreover, the protonaffinity of the amide nitrogen atom is increased by asecond substituent. Finally, an excess of amide groupsis present in the D3A4 oligomers compared to one allylicmoiety.

Upon ESI, the m/z 808 [D3A3 2 H2O 1 H]1 ion is thebase peak of the [D3A3 2 mH2O 1 H]1 cluster (Figure2 and Table 2) and carries 69% of the ESI ion current ofthe D3A3 end-group distribution, whereas m/z 790[D3A3 2 2H2O 1 H]1 constitutes 64% of the MALDI

end-group distribution (see Table 3). The presence ofD3A3 oligomers in the polyesteramide chemical compo-sition is revealed by ESI formation of m/z 826 [D3A3 1H]1 ions. m/z 826 is not observed in the MALDIspectrum. The FD determined end-group compositionis somewhere between the MALDI and ESI one. Forma-tion of m/z 905 [D3A4 2 2H2O 1 H]1 indicative ofdouble water losses from the end-groups in the course

Figure 2. ESI mass spectrum of a hyperbranched polyester amide: D denotes a hexahydrophthalicacid monomer and A indicates a diisopropanol amine.

Table 3. Relative end-group composition of the D3A3 andD3A4 oligomers (normalized to 100%)

Ion structure MALDI FD ESI

m/z 790 [D3A3 2 2H2O 1 H]1 64 44 16m/z 808 [D3A3 2 H2O 1 H]1 36 45 69m/z 826 [D3A3 1 H]1 — 11 15m/z 905 [D3A4 2 2H2O 1 H]1 16 18 3m/z 923 [D3A4 2 H2O 1 H]1 72 38 21m/z 941 [D3A4 1 H]1 12 44 76 Figure 3. PSD/MALDI-TOF mass spectrum of collisionally acti-

vated m/z 654 HPEA [D2A3 2 H2O 1 H]1 cations.

223J Am Soc Mass Spectrom 2000, 11, 218–227 IN-SOURCE DECAY OF HYPERBRANCHED POLYMERS

of the synthesis is negligible according to the ESI results(see Table 3).

These observations practically hold for the entiredata set (Table 2). In conclusion, there are pronounceddifferences between the MALDI, FD, and ESI spectra.The most abundant peaks in a cluster of [DnAn11 22H2O 1 H]1, [DnAn11 2 H2O 1 H]1, and [DnAn11 1H]1 ions with the same degree of oligomerization n inthe ESI-MS spectrum are those due to the m/z[DnAn11 1 H]1 ions. The FD peak intensities due toboth [DnAn11 2 H2O 1 H]1 and [DnAn11 1 H]1 ionsare almost equal, whereas the m/z [DnAn11 1 H]1 peakis almost absent in the MALDI spectrum (Table 2).

For the DnAn clusters similar results were obtained.Protonated DnAn molecules, which are absent in theMALDI spectrum, are formed and detected upon FDand ESI of the sample. Apparently, a population ofDnAn oligomers that have not lost a water molecule bya cyclization reaction is present in the chemical compo-sition distribution of the hyperbranched polyesteramides. These experimental observations are indicativefor the fact that the end-groups are lost by ISD uponMALDI and not during the synthesis of the polyester-amides. This assertion is further corroborated byMALDI/PSD experiments.

Post-Source Decay MALDI-MS/MS

The components of the chemical composition distribu-tion were characterized by PSD of mass selected ionswith and without collision gas. The ion current of the[DnAn11 1 H]1 and [DnAn 1 H]1 is too low to obtainPSD spectra of these ions. PSD spectra of [DnAn11 2mH2O 1 H]1 and [DnAn 2 mH2O 1 H]1 were readilyobtained. As stated above there is a structural analogy

between the HPEA’s and peptides concerning the pres-ence of the amide bonds. The DA dimer is in thisanalogy considered as an amino acid. In order todeduce fragmentation rules for the HPEAs we used asa starting hypothesis well established gas-phase pep-tide ion chemistry. Definite proof of detailed mecha-nisms could in this study not be obtained by lack ofsuitable labeled compounds. A detailed MALDI-MS/MS and ESI-MS/MS study of the hyperbranchedpolyesteramide chemical composition distribution willbe published elsewhere.

The MALDI PSD spectrum of mass selected m/z 654[D2A3 2 H2O 1 H]1 in the presence of collision gas isgiven as a typical example in Figure 3. As a startinghypothesis we assume that the m/z 654 cation popula-tion consists of D2A3 oligomers which have lost an OHend-group by water elimination from an isopropanolmoiety during the synthesis leading to an allylic unsat-uration in one of the secondary amide side chains.Protonation at the amide bond followed by collisionallyinduced loss of neutral unsaturated isopropanol-1-pro-pene amine (A 2 H2O) or loss of diisopropanol amine(A) yields B2 cations at m/z 539 and m/z 521 (eq 2),respectively (Scheme 3):

[D2A3 2 H2O 1 H]1

3 [D2A2 2 H2O 1 H]1 1 A 2 H2O (2)

3 [D2A2 2 2H2O 1 H]1 1 A

The m/z 252 and 270 B1 ions are formed by unimolecularlosses of ADA and (ADA 2 H2O) neutrals, respectively(vide Scheme 3). B1 formation is, in contrast to peptideion chemistry, possible because the positive charge on

Scheme 3

224 MUSCAT ET AL. J Am Soc Mass Spectrom 2000, 11, 218–227

the carbonyl C-atom ions is stabilized by intramolecularquarternary ammonium ion formation, i.e., a diisopro-panol hexahydrophthalimide cation [30]. This stabiliza-tion readily hinders loss of carbon monoxide yieldingA1 ions. The B2 ions are stabilized by oxazolonium ionformation. The m/z 270 B1 and m/z 539 B2 ions areisomers of cyclic [DA 2 H2O 1 H]1 and cyclic[D2A2 2 H2O 1 H]1 cations, respectively. Higher mo-lecular mass DnAn 2 H2O oligomers show similar uni-molecular fragmentation reactions upon collisional ac-tivation. Although a PSD spectrum of the m/z 672[D2A3 1 H]1 has not been obtained, we generalize Bion formation (eq 2) as a universal reaction channel forcollisionally activated [DnAn11 1 H]1 oligomers (eq 3):

[DnAn11 1 H]13 [DmAm 2 H2O 1 H]1

1 Dn2mAn2m11 (3)

Another reaction mode involves dissociation of theester bond between a diisopropanolamine and one ofthe diacid carboxylic groups (Scheme 4). This dissocia-tion reaction produces m/z 385 [DA2 2 H2O 1 H]1 orm/z 367 [DA2 2 2H2O 1 H]1 cations dependent on thesite of the unsaturation and DA 2 H2O or DA neutrals(eq 4), respectively:

[D2A3 2 H2O 1 H]1

3 [DA2 2 2H2O 1 H]1 1 DA 2 H2O (4)

3 [DA2 2 H2O 1 H]1 1 DA

Formation of this ion proceeds by intramolecular trans-fer of the ionizing proton from the amide bond to thecarboxyl O atom of the ester bond. A mesomeric form of

the latter corresponds to an intermediate where thecharge is localized on the O atom between the carbonylC atom and the isopropanol moiety. Neighboring-group participation of the amide carbonyl O atomenhances an anchimeric assisted intramolecular nucleo-philic substitution reaction leading to displacement ofthe DA neutral with concomitant ring closure yielding astable oxazolinium ion, further denoted as OX (seeScheme 4). The reaction is driven by formation of thethermochemically stable oxazolonium cation. The m/z385 OX1 ion is an isomer of the linear [DA2 2 H2O 1H]1 cation. Higher molecular mass DnAn11 2 H2Ooligomers show similar oxazolonium cation formationupon collisional activation. We generalize reaction 4 asa reaction channel for collisionally activated [DnAn11 1H]1 oligomers (eq 5):

[DnAn11 1 H]13 [DmAm11 2 H2O 1 H]1

1 Dn2mAn2m (5)

Reaction 2 and 4 are also observed without collision gas.More in general, MALDI/PSD spectra of mass selectedprotonated DnAn11 oligomers without collision gasreveal that a substantial rapid metastable unimolecularfragmentation of the protonated oligomers within themass spectrometric time scale takes place. The PSDresults also show that metastable reactions proceed viapathways leading to lower oligomers minus a watermolecule by B and OX ion formation (Schemes 3 and 4).For protonated DnAn oligomers similar conclusions, i.e.,metastable fragmentation to lower oligomers, are ob-tained (data not shown). The occurrence of rapid meta-stable decay post-ion extraction in combination with theMALDI, FD, and ESI comparison of end-group distri-

Scheme 4

225J Am Soc Mass Spectrom 2000, 11, 218–227 IN-SOURCE DECAY OF HYPERBRANCHED POLYMERS

butions provide strong evidence that metastable decayalso occurs in the ion source prior to ion extraction(ISD). Although, thermal loss of water from desorbedneutral followed by MALD ionization cannot be ex-cluded.

This also means that m/z 654 [D2A3 2 H2O 1 H]1

ions can originate by DA loss from m/z 940 [D3A4 1H]1, D2A2 loss from m/z 1209 [D4A5 1 H]1 cations, etcin the ion source. Our initial assumption that the m/z654 ion population consists of D2A3 oligomers whichhave an allylic unsaturation in one of the secondaryamide isopropanol side chains has to be extended. Them/z 654 population is composed of m/z 654 ions with anunsaturation already present prior to MALDI and m/z654 oxazolonium ions formed via eq 5 from higheroligomers by ISD.

Conclusions

MALDI TOF-MS characterization of hyperbranchedpolyesteramides gives at first instance the impressionthat part of the OH end-groups is lost in the syntheses.The MALDI, FD, and ESI derived end-group composi-tions of HPEA show that end-groups which are appar-ently not a structural entity of oligomers in the MALDIdistribution are present in the FD and ESI distributions,thus are present on target prior to MALDI ionization.Furthermore, metastable reaction paths are open fordissociation of higher oligomers to lower oligomersminus a water molecule. These observations providestrong evidence for ISD of protonated hyperbranchedpolyesteramides. ISD causes an actual end-group loss inthe ion source of the mass spectrometer, i.e., higheroligomers dissociate into lower oligomers minus awater molecule within the mass spectrometric timescale, upon MALD ionization.

Characterization and chemical composition analysisof polymers by use of MALDI-TOF-MS is a feasible andvaluable method, because it provides oligomer-re-solved information. However, MALDI induced frag-mentation of the oligomer ions in the ion source prior toion extraction can affect the distribution, leading towrong conclusions concerning the chemical composi-tion distribution and the end-group distribution.

AcknowledgmentThe authors gratefully acknowledge the management of DSMResearch for the permission to publish this work.

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