Review An Organic ’s Guide to Spectrometric Elucidation

Arnold Steckel 1 and Gitta Schlosser 2,*

1 Hevesy György PhD School of , ELTE Eötvös Loránd University, Pázmány Péter sétány 1/A, 1117 Budapest, Hungary; [email protected] 2 Department of , ELTE Eötvös Loránd University, Pázmány Péter sétány 1/A, 1117 Budapest, Hungary * Correspondence: [email protected]

 Received: 16 January 2019; Accepted: 8 February 2019; Published: 10 February 2019 

Abstract: Tandem is an important tool for structure elucidation of natural and synthetic organic products. Fragmentation of odd (OE+) generated by electron (EI) was extensively studied in the last few decades, however there are only a few systematic reviews available concerning the fragmentation of even-electron ions (EE+/EE−) produced by the currently most common ionization techniques, (ESI) and atmospheric pressure (APCI). This review summarizes the most important features of tandem mass spectra generated by collision-induced dissociation fragmentation and presents didactic examples for the unexperienced users.

Keywords: ; MS/MS fragmentation; collision-induced dissociation; CID; ESI; structure elucidation

1. Introduction (EI), a hard ionization technique, is the method of choice for analyses of small (<1000 Da), nonpolar, volatile compounds. As its name implies, the technique involves ionization by with ~70 eV . This energy is high enough to yield very reproducible mass spectra with a large number of fragments. However, these spectra frequently lack the type molecular ions (M+) due to the high transferred to the precursors [1]. This possible lack of molecular weight information is one of the greatest limitations of this ionization method. Soft ionization techniques, including electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are nowadays routinely used to ionize thermally labile and at least moderately polar organic analytes. These methods usually yield ions with no unpaired electrons (even-electron ions, EE) and the resulting [M + H]+ species are referred to as protonated molecules. Note that use of the term quasi-molecular ions is discouraged [2]. These ions possess low internal energy, therefore fragmentation in a single stage MS experiment is negligible [3–8]. This way, the intact accurate mass for sensitive, fragile compounds and large as well as their detailed could be determined, even when high pressure is used as a prefractionation method. As the application of electrospray ionization is far more widespread than any other ionization type, only this technique will be reviewed herein.

2. Choosing the Precursor for Fragmentation ESI methodology can be used in either positive (ES+) and negative ion mode (ES−), depending on the affinity of the compound. In ES+, only analytes with at least one positive , and in ES−, only analytes with at least one negative elementary charge, can be detected. Molecules

Molecules 2019, 24, 611; doi:10.3390/molecules24030611 www.mdpi.com/journal/molecules Molecules 2019, 24, x FOR PEER REVIEW 2 of 11 Molecules 2019, 24, 611 2 of 11 2. Choosing the Precursor Ion for Fragmentation possessingESI basic methodology characteristics can be couldused in beeither easily positive ionised (ES+) in and ES+ negative via making ion mode adducts (ES−), depending with proton(s), while moleculeson the proton having affinity acidic of the functional compound. groups—and In ES+, only lackinganalytes basicwith at ones—typically least one positive give elementary better spectra charge, and in ES−, only analytes with at least one negative elementary charge, can be detected. in ES−. Optimal ionization mode selection can be based on the Brønsted-Lowry - theory [9]. Molecules possessing basic characteristics could be easily ionised in ES+ via making adducts with It shouldproton(s), be emphasized while molecules that only having electrospray-compatible acidic functional groups—and lacking (e.g., basic , ones—typically , give , )—containingbetter spectra in ES 0.1%−. Optimal formic ionization or acetic mode acid toselection enhance can —or be based on the Brønsted-Lowry acid- can be used forbase dissolving theory [9]. It samples should be during emphasized sample that preparation. only electrospray-compatible Solvents are highly solvents recommended (e.g., acetonitrile, to be of LC-MSwater, grade. methanol, Doubly ethanol)—containing distilled water should 0.1% be formic used or for acetic aqueous acid to enhance protonation—or to effectively minimize solvent the intensitymixtures of can be adducts used for in thedissolving spectra. samples It is also during advisable sample to usepreparation. low-binding, Solvents high-quality are highly plastic samplerecommended tubes to decrease to be of the LC-MS otherwise grade. Doubly always distilled present water plasticizer should contamination.be used for aqueous solutions to effectively minimize the intensity of sodium adducts in the spectra. It is also advisable to use low- The type of EE ions generated in the source are usually singly or multiply protonated [M + zH]z+ binding, high-quality plastic sample tubes to decrease the otherwise always present plasticizer − − − ions incontamination. ES+, while singly deprotonated [M H] ions are dominant in ES . Multiply protonated species areThe usually type of formed EE ions fromgenerated large in molecules the source are (e.g., usually ), singly or or multiply ones having protonated multiple [M + z favorableH]z+ sites forions protonation. in ES+, while Thesesingly deprotonated ions are fragmented [M − H]− ions more are easily dominant due in to ES charge−. Multiply repulsion; protonated however, fragmentsspecies are are often usually not formed too informative. from large molecules If the (e.g., polymers), is large enough,or ones having it will multiple be represented favorable by an envelope-shapedsites for protonation. distribution These of ions peaks. are fragmented more easily due to charge repulsion; however, Chargefragments distributions are often not can too informative. be altered byIf the using molecu buffers.le is large Adduct enough, formation it will be represented could also by take an place envelope-shaped distribution of peaks. depending on the Lewis basicity of the compound in the presence of NH +, Na+ and K+ or if Li+ Charge distributions can be altered by using buffers. Adduct formation could4 also take place or Ag+ is intentionally added to the sample [10,11]. Note that these ions should be referred to as depending on the Lewis basicity of the compound in the presence of NH4+, Na+ and K+ or if Li+ or Ag+ cationizedis intentionally molecules added as the toterms the sample quasi-molecular [10,11]. Note th ionat andthese pseudo-molecular ions should be referred ion to are as discouragedcationized [2]. Thesemolecules adduct ions as the usually terms quasi-molecular give no fragmentation ion and pseudo-molecular other than the lossion are of thediscouraged cation, but[2]. These for special compoundsadduct (e.g.,ions usually carbohydrates), give no fragmentation their fragmentation other than the could loss of be the also cation, significantly but for special different compounds from that of [M(e.g., + H] +carbohydrates),ions, giving their useful fragmentation structural informationcould be also [significantly12]. Ammonium different acetate from that or of bicarbonate [M + H]+ are usuallyions, used giving for useful adduct structural formation information typically [12]. in Ammonium 5 mM acetate or bicarbonate (Figure1). are It usually is worth used noting for adduct formation typically in 5 mM concentration (Figure 1). It is worth noting that additives with that additives with high ionization efficiency (e.g., tertiary or quaternary amines) and ones causing high ionization efficiency (e.g., tertiary or quaternary amines) and ones causing significant significant suppression of signal intensity (e.g., phosphate buffers, sodium , trifluoroacetate) suppression of signal intensity (e.g., phosphate buffers, , trifluoroacetate) should be shouldavoided. be avoided. In ES− In, adduct ES−, adductformation formation could also could happen; also anionized happen; anionizedmolecules may molecules be useful may in bethe useful in thedetection of of some some compounds compounds (e.g., (e.g., carbohydrates, carbohydrates, flavonoids). flavonoids).

FigureFigure 1. Single 1. Single stage stage mass mass spectrum of of trehalose, trehalose, aa disaccharide found found e.g. e.g.,, in insects. in insects. Due Dueto the to lack the lack of basicof residuesbasic residues in the in the molecule, molecule, [M [M + + H]H]+ ionsions could could not not be bedetected detected if the if sample the sample was dissolved was dissolved in in acetonitrile-wateracetonitrile-water 1:1 1:1 (v(v//v) solventsolvent , mixture, containing containing 0.1% 0.1 acetic% acetic acidacid (spectrum (spectrum not shown). not shown). However, using 5 mM of ammonium acetate in water resulted in the above spectrum. Although

absolute intensity is still low, the ammonium adduct of trehalose and its cationized dimer could also be detected. Also, a possible in-source fragmentation product can be seen at m/z 325 displaying a neutral loss of water (◦) and (*). Note the peaks corresponding to m/z 296.1 and m/z 338.4 are very + + likely the [M + NH4] ion of dibutyl phtalate and [M + H] ion of erucamide, respectively, which are common plasticizers leaked from plastic sample tubes. Molecules 2019, 24, x FOR PEER REVIEW 3 of 11

However, using 5 mM of ammonium acetate in water resulted in the above spectrum. Although absolute intensity is still low, the ammonium adduct of trehalose and its cationized dimer could also be detected. Also, a possible in-source fragmentation product can be seen at m/z 325 displaying a neutral loss of water (°) and ammonia (*). Note the peaks corresponding to m/z 296.1 and m/z 338.4 + + Moleculesare 2019very, 24likely, 611 the [M + NH4] ion of dibutyl phtalate and [M + H] ion of erucamide, respectively,3 of 11 which are common plasticizers leaked from plastic sample tubes.

If the the analyte analyte is is prone prone to to complex complex formation formation or or is is highly highly concentrated, concentrated, [2M [2M + H] + H]+ or+ [2Mor [2M − H]−− ionsH]− ionscould could also be also present be present in the in full the mass full mass spectra. spectra. It is Italso is alsocommon common to detect to detect M+ ions M+ ionsusing using soft ionizationsoft ionization methods methods if the ifcompound the compound already already had a fixed had acharge fixed chargebefore ionization before ionization (Figure 2). (Figure Typical2). examplesTypical examples are quaternary are quaternary ammonium ammonium salts. The salts. distribution The distribution of charge ofvia charge conjugation via conjugation also enhances also theenhances stability the of stability the protonated/deprotonated of the protonated/deprotonated ion as e.g., ion in ascase e.g., of in compounds case of compounds with a dienone with a dienonemoiety. Ifmoiety. a given If acompound given compound (e.g., ketone (e.g., ketones,s, aldehydes, aldehydes, alcohols) alcohols) cannot cannot be detected be detected in either in either mode mode or fragmentationor fragmentation efficiency efficiency needs needs to tobe be enhanced, enhanced, one one could could use use derivatizati derivatizationon agents agents [[13].13]. These compounds are usuallyusually basicbasic toto enhanceenhance ionizationionization efficiency. efficiency. The The limitation limitation of of this this approach approach is is that that it itcannot cannot be be used used for for totally totally unknown unknown compounds. compounds.Another Another wayway isis toto useuse aa differentdifferent ionization type such as APCIAPCI [[14]14] oror atmospheric atmospheric pressure pressure photoionization (APPI), (APPI) which, which may may be be more more applicable applicable for forless less polar polar compounds compounds (e.g., (e.g., lipids) lipids) [15]. [15]. Note Note that molecularthat molecular ions (Mions+) could(M+) could be also be observed also observed using APCIusing orAPCI APPI or [APPI14–16 [14–16].].

Figure 2. Illustration of Field’s rule on thiamine (vitamin B ) in positive mode (ES+). Note that Figure 2. Illustration of Field’s rule on thiamine (vitamin B1) 1in positive mode (ES+). Note that the + + precursorthe precursor in blue in blueis not is a protonated not a protonated thiamine thiamine molecule molecule [M + H] [M+, but + H]simply, but an simply M+, as the an compound M , as the compound intrinsically has a fixed positive charge. Upon collision-induced dissociation, it decomposes intrinsically has a fixed positive charge. Upon collision-induced dissociation, it decomposes via two via two different pathways (orange and green colors) to yield product ions (upper structures) and different pathways (orange and green colors) to yield product ions (upper structures) and neutral neutral molecules (lower structures). The pathway in orange represents an inductive cleavage (see molecules (lower structures). The pathway in orange represents an inductive cleavage (see also also Scheme1), while the one in green indicates an acyclic rearrangement competitive with Scheme 1), while the one in green indicates an acyclic hydrogen rearrangement competitive with the the former. Only charged fragments could be detected (upper structures), and their intensities are former. Only charged fragments could be detected (upper structures), and their intensities are proportional to their neutral forms’ proton affinity. Note that the fragments could be represented proportional to their neutral forms’ proton affinity. Note that the fragments could be represented with with multiple other (more stable) structures; here, only one of each is depicted due to multiple other (more stable) resonance structures; here, only one of each is depicted due to didactic didactic reasons. reasons.

3. Performing MS/MS Experiments To obtain structural information from an EE ion, an MS/MS (tandem mass spectrometric) experiment is needed either in a collision cell or in an instrument featuring an analyzer. Protonated initial molecules selected for fragmentation are called precursor ions, while their

Molecules 2019, 24, 611 4 of 11 Molecules 2019, 24, x FOR PEER REVIEW 6 of 11

Scheme 1.1. RepresentationRepresentation of of the the main main noncyclic noncyclic fragmentation fragmentation pathways pathways characteristic characteristic to even-electron to even- ionelectron fragmentation ion fragmentation in ES+ mode. in ES+ Green mode. arrows Green indicatearrows indicate inductive inductive cleavages, cleavages, while blue while ones blue denote ones acyclicdenote βacyclic-H-rearrangements. β-H-rearrangements. The molecule The molecule of interest of must interest contain must at leastcontain one at functional least one group functional prone togroup be protonated. prone to be Xprotonated. denotes -NH, X denotes -O- or -S--N groups,H, -O- or while -S- groups, Y denotes while -CH Y2 denotes-, C=O, -SO-CH22-,ones C=O, so -SO that2 theones general so that fragmentationthe general fragmentation routes for amines, routes (sulfon)amides,for amines, (sulfon)amides, ethers, esters, ethers, thioesters esters, are thioesters given here. are Moleculesgiven here. featuringMolecules groups featuring not groups covered not by covered the scheme by the above, scheme like above, alcohols like or alcohols carboxylic or carboxylic are proneacids are to loseprone water to orlose water dioxide/formic or / acidformic upon collision-induced acid upon collision-induced dissociation respectively.dissociation Ketonesrespectively. usually Ketones dissociate usually to yield dissociate acylium to ions, yield while acylium simple ions, aldehydes while simple are often aldehydes cannot be are detected often withoutcannot be derivatization. detected without Expressive derivatization. and more Expressive specific examples and more are collectedspecific examples by Niessen are et collected al. [2] (p. 84;by orNiessen 94–101 et for al. general[2] (p. 84; routes or 94–101 of fragmentation for general routes for specific of fragmentation compound classes). for specific compound classes). 3. Performing MS/MS Experiments Fragmentation of even-electron ions usually follow the so-called even-electron rule [36,37]. BasedTo on obtain the even-electron structural information rule, the formation from an of EE ions ion, with an no MS/MS unpaired (tandem electrons mass are spectrometric)favored upon experimentfragmentation, is neededalong with either loss in aof collision neutral cell molecules or in an from instrument the precursor, featuring instead an ion trapof homolytic analyzer. Protonatedcleavages which initial moleculesare rare amongst selected forthis fragmentation type of ions. areEliminations called precursor of neutral ions, whilemolecules their fragmentscould be arecharacteristic referred to (selective as product or ions.specific) The to terms certain mother types and of compounds. daughter ions For are a discouragedvery thorough [2 ],and however, useful theylist on are characteristic still widely neutral used. Bothlosses, in see traps Niessen and collision et al., 2017 cells, [2]. the analyte ions generated by the ion sourceHeterolytic are excited cleavage by collisions is often with referred inert to as indu e.g.,ctive N2, cleavage Ar or He—depending and can be observed on the for instrument both OE type—toand EE ions. impart The enough inductive energy cleavage inducing of the decomposition carbon-heteroatom processes bond [17]. is It a should CMF alsoleading be noted to an that EE fragmentationfragment. Typical in theinductive cleavage could products occur with are re considerablepresented in extent Figure for 3 for fragile protonated compounds bisoprolol. or by alteringA competitive source parameters.fragmentation This pathway could be exploitedwith inductive in case ofcleavage using quadrupole of EE ions ion is trapsthe whereacyclic useful β-H- fragmentsrearrangement could (a be CRF) missing and incould standard be deducted MS/MS from spectra an dueinductive to a technical cleavage limitation where a calledproton low-mass at the β- cut-off.carbon migrates Another to way the for other the inductionfragment. ofThus, in-source in soft fragmentation ionization, heterolytic where there cleavage is no secondalso include mass analyseracyclic β- (e.g.,H-rearrangement. single quadrupole For both instruments) pathways, is performingthe sum of conethe two fragments fragmentation/declustering is equal to M + 2H+, potentialand the relative fragmentation. abundance These of them methods is determined could only by be the used Field’s however rule. Inductive if the compound cleavage is and purified acyclic or MSβ-H-rearrangement is hyphenatedwith are two HPLC. of the Investigation most important of the cleavage fragmentation types in processesthe dissociation in case of of EE EE+ ions. ions are usually carried out using various methods, such as theoretical calculations, stable labelling, H/D exchange [18] and MSn experiments on instruments capable of high resolution and high mass accuracy (HRAM-MS/MS). and Q-TOF instruments are usually the best method of choice for these purposes as these are the most widely applicable types. When calculating molecular and comparing them to experimentally obtained values, monoisotopic masses (often called exact masses in molecule editor programs) must be used if the instrument is capable of HRAM-MS/MS. If MS/MS is carried out in TOF/TOF instruments (keV imparted to precursors) or is coupled to ion

Molecules 2019, 24, 611 5 of 11 mobility spectrometry, discrimination between constitutional or diastereomers respectively, is possible [19–21]. These methods are mostly used in the analyses of biomolecules. In the following, the general rules for fragmentation processes of protonated molecules will be outlined. The rules will be applied on some examples to demonstrate their usefulness. There is far less information on the fragmentation processes of deprotonated molecules in the literature, so we will restrict the observations for ES+ mode. One reason for this is that the formation of deprotonated molecules is usually limited to compounds possessing acidic or compounds that could generate such ones by tautomerism. Other reasons include the poor understanding of fragmentation processes and technical difficulties. The signal intensity for ES− is usually lower, therefore whenever possible, use of ES+ is recommended. However, ES− is of high importance e.g., for the characterization of flavonoids [6], oligosaccharides [22], carboxylic acids [23,24], sulfonamides [25], oligonucleotides [26] and rarely [27].

4. General Fragmentation Characteristics of Protonated Molecules The fragmentation characteristics of an [M + zH]z+ type protonated molecule and an M+ radical type molecular ion is significantly different (Table1).

Table 1. Comparison of the fragmentation of odd and even-electron precursors upon fragmentation.

Odd Electron Precursor (OE) Even-Electron Precursor (EE) Ionization EI ESI, MALDI, APCI, (APPI) 1 Type of precursor M+ [M + zH]z+, [M + C]+, [M − H]−, [M + A]− rule   RDBE 2 integer half-integer Type of fragments OE EE Prediction of fragment Stevenson’s rule Field’s rule abundance Preferred cleavage type homolytic heterolytic 1 ESI: electrospray ionization, MALDI: matrix-assisted desorption/ionization, APCI: atmospheric pressure chemical ionization, APPI: atmospheric pressure photoionization. 2 RDBE: ring and equivalent. RDBE NY NZ should be calculated as NX − 2 + 2 + 1 where X = C, Si; Y = H or , Z = N, P ([1], p. 28, [28]) Note that and are not included in this formula and all atoms are considered with their lowest valence state. Despite other attempts to calculate the degree of unsaturation, there is none that could be used without limitation.

OE ions tend to fragment at more random sites, while even-electron ions usually produce less but more stable fragments by cleavages at the thermochemically least stable bonds. It is also possible to form an odd-electron ion from an ESI-generated multiply charged even-electron ion precursor with methods of electron capture dissociation (ECD) [29] or dissociation (ETD) [30] which nowadays have widely-used application in of peptides and other biomolecules. It is supposed that the mobile proton hypothesis could also play an important role in the fragmentation of protonated small organics, as previously described for peptides in the literature [31,32]. Based on this hypothesis, protons occupy the favored basic sites of the molecules via soft ionization in the source. Afterwards, excitation in the collision cell or in the trap results in proton migration throughout the structure. When protons reach a specific position causing thermal instability for the system, the ion decomposes leading to two fragments: one of them will carry the original charge, the other will be eliminated as a neutral molecule. According to some authors, the neutral molecule and the charged ion could also form an ion/neutral complex [33] and the to-be-fragments could successfully compete for the sequestration of the proton yielding to complementary fragment ions in the MS/MS spectra. The most important qualitative rule to predict which fragment will retain the charge and how abundant will be in the spectrum is to investigate the proton affinity of their neutral counterparts. The higher is the proton affinity of a neutral, the higher is the intensity of its charged form in the spectrum. This is called Field’s rule [34] and is depicted in Figure2. Molecules 2019, 24, 611 6 of 11

All these processes are unimolecular; therefore, fragments could only contain elements that could already be found in the precursor as well. The presence of elements with special isotope distribution like , , certain transition or intentionally introduced stable could also contribute to an easier structure elucidation. The so-called ‘nitrogen rule’ is routinely applied for EI spectra. It states that a singly charged OE precursor/fragment ion with an odd nominal m/z contains odd number of N atoms; an ion with an even nominal m/z contains even number of N atoms. The nitrogen rule is also applicable for soft ionization methods [1,28] (Table2): an odd nominal m/z of an EE ion indicates an even number of N atoms, and an even nominal m/z of an EE ion indicates an odd number of N atoms.

Table 2. The extension of nitrogen rule to even-electron ions ([1], p. 38). Note that the N rule is only applicable for singly charged ions containing solely H, C, N, O, Hlg, P and S atoms and is only reliable < m/z 500 [28].

Odd m/z Even m/z OE 1 odd #N even #N EE 2 even #N odd #N 1 odd-electron precursor/fragment ion. 2 even-electron precursor/fragment ion.

Types of Cleavages and Reaction Routes The fundamental fragmentation pathways could be classified as radical site-induced fragmentation, charge-induced fragmentation and charge-remote fragmentation according to McLafferty [1]. For odd-electron ions, all these routes are allowed, in contrast to even-electron ions where only the last two are possible. For depiction of mechanisms, curved double-headed arrows are used for the movement of an electron pair while single-headed ones are for displaying the motion of a single electron. Bond cleavages upon the decomposition of an ion can be sorted as either homolytic or heterolytic. Another type of characterization of the fragmentation processes is based on the retention of the charge. In case of charge retention fragmentations (CRF) the charge is retained on the possessing the charge initially, while in case of charge migration fragmentations (CMF) charge is transferred to one of the atoms in the other fragment. The most fundamental fragmentation pathways of positively charged even-electron ions are summarized in Scheme1. For more specific examples, see Figures3–6 (from the authors’ own works) or Niessen et al. [2,35]. Fragmentation of even-electron ions usually follow the so-called even-electron rule [36,37]. Based on the even-electron rule, the formation of ions with no unpaired electrons are favored upon fragmentation, along with loss of neutral molecules from the precursor, instead of homolytic cleavages which are rare amongst this type of ions. Eliminations of neutral molecules could be characteristic (selective or specific) to certain types of compounds. For a very thorough and useful list on characteristic neutral losses, see Niessen et al., 2017 [2]. Heterolytic cleavage is often referred to as inductive cleavage and can be observed for both OE and EE ions. The inductive cleavage of the carbon-heteroatom bond is a CMF leading to an EE fragment. Typical inductive cleavage products are represented in Figure3 for protonated bisoprolol. A competitive fragmentation pathway with inductive cleavage of EE ions is the acyclic β-H-rearrangement (a CRF) and could be deducted from an inductive cleavage where a proton at the β-carbon migrates to the other fragment. Thus, in soft ionization, heterolytic cleavage also include acyclic β-H-rearrangement. For both pathways, the sum of the two fragments is equal to M + 2H+, and the relative abundance of them is determined by the Field’s rule. Inductive cleavage and acyclic β-H-rearrangement are two of the most important cleavage types in the dissociation of EE+ ions. Molecules 2019, 24, 611 7 of 11 Molecules 2019, 24, x FOR PEER REVIEW 7 of 11

Molecules 2019, 24, x FOR PEER REVIEW 7 of 11

FigureFigure 3. Tandem 3. Tandem mass spectrum of of bisoprolol, bisoprolol, a cardioselective β-blocker.β-blocker. The Theinitial initial site of site of protonation is the basic sp33 N. Without this moiety, the molecule cannot be detected. After collision- protonationFigure 3. is Tandem the basic mass sp spectrumN. Without of bisoprolol, this moiety, a cardioselective the molecule β-blocker. cannot The be initial detected. site of After induced activation, the precursor ion [M + H]+ can+ be represented by two structures. All two collision-inducedprotonation is activation, the basic sp the3 N. precursor Without this ion moiety [M +, H]the moleculecan be represented cannot be detected. by two After structures. collision- All two fragments of the precursor ion are a result of inductive cleavages in concordance with Scheme 1, green fragmentsinduced of the activation, precursor the ion precursor are a result ion [M of inductive+ H]+ can cleavagesbe represented in concordance by two structures. with Scheme All two1, green arrows (in this case, X = O, Y = CH2). Note that these fragments not necessarily represent the most arrowsfragments (in this of case, the precursor X = O, Y ion = CH are 2a). result Note of thatinduct theseive cleavages fragments in concordance not necessarily with Scheme represent 1, green the most stable resonance structures. stablearrows resonance (in this structures. case, X = O, Y = CH2). Note that these fragments not necessarily represent the most stable resonance structures. Homolytic cleavages include σ- (mostly for OE) and α-cleavage (only for OE). The role of σ-bond Homolytic cleavages include σ- (mostly for OE) and α-cleavage (only for OE). The role of σ-bond homolyticHomolytic cleavage cleavages in EE+ include fragmentation σ- (mostly processes for OE) are and limited α-cleavage to certain (only ty forpes OE). of compounds The role of σ having-bond homolytica carbon-heteroatom cleavage in EE+ bond fragmentation resulting in processesthe loss of areradical limited species to certainsuch as typesCl˙, NO of2˙ compounds, ˙CH3, CH3O having˙ homolytic cleavage in EE+ fragmentation processes are limited to certain types· of compounds· · having· a carbon-heteroatom[3,27]. For this type bond of violation resulting of ineven-electron the loss of rule radical, see Figure species 4. suchThese as losses Cl , NOof radicals2 , CH are3, CHusually3O [3,27]. a carbon-heteroatom bond resulting in the loss of radical species such as Cl˙, NO2˙, ˙CH3, CH3O˙ For this[3,27].competitive type For of this violationwith type the of loss violation of of even-electron their of non-radicaleven-electron rule, counterparts rule see, see Figure Figure e.g.,4. 4.HCl. These These losses losses of of radicals radicals are usually are usually competitivecompetitive with with the lossthe loss of theirof their non-radical non-radical counterparts counterparts e.g., e.g., HCl. HCl.

Figure 4. In-source fragmentation of protonated tianeptine, marketed as a nootropic ‘dietary supplement’ in the USA. Note that m/z 213 and 193 correspond to fragments that violate the above- FigureFigure 4. In-source 4. In-source fragmentation fragmentation ofof protonatedprotonated tian tianeptine,eptine, marketed marketed as a as nootropic a nootropic ‘dietary ‘dietary mentioned even-electron rule. Elimination of SO2 is a charge-remote neutral loss characteristic to supplement’supplement’ in the in the USA. USA. Note Note that mm/z/ 213z 213 and and193 correspond 193 correspond to fragments to fragments that violate that the above- violate the sulfonamides [38], sulfonic acids and aromatic sulfoxides. The mechanisms for losses and above-mentionedmentioned even-electron even-electron rule. rule. Elimination Elimination of SO of2 is SO a charge-remoteis a charge-remote neutral neutralloss characteristic loss characteristic to rearrangement reactions are often not trivial but rules2 known from regarding sulfonamides [38], sulfonic acids and aromatic sulfoxides. The mechanisms for losses and to sulfonamidesstability of ions [38 could], sulfonic be successfully acids andapplied. aromatic sulfoxides. The mechanisms for losses and rearrangementrearrangement reactions reactions are are often often not not trivial trivial butbut rules known known from from organic organic chemistry chemistry regarding regarding stability of ions could be successfully applied. stability of ions could be successfully applied.

It is often required for atoms and bonds to be rearranged either through a six-membered cyclic transition state or through ion/neutral complexes prior to fragmentation. Most common rearrangement types for protonated molecules include retro (hetero)ene reactions and retro Diels-Alder Molecules 2019, 24, 611 8 of 11 Molecules 2019,, 24,, xx FORFOR PEERPEER REVIEWREVIEW 8 of 11

It is often required for atoms and bonds to be rearranged either through a six-membered cyclic reaction. DidacticIt is often examples required for for atoms these and types bonds are to reviewedbe rearranged by either Demarque through eta six-membered al. [6]. A retro-heteroene cyclic transition state or through ion/neutral complexes prior to fragmentation. Most common reaction couldrearrangement be considered types for as protonated the even-electron molecules incl counterpartude retro (hetero)ene of McLafferty reactions rearrangement. and retro Diels- In case of retro-Diels-AlderAlder reaction. reaction, Didactic the examples six-member for these ring types with are a double reviewed bond by Demarque is given inet theal. [6]. first A place,retro- there is no needheteroene for the preformation reaction could of suchbe considered structures. as Thesethe even-electron three reactions counterpart along with of theMcLafferty elimination of rearrangement. In case of retro-Diels-Alder reaction, the six-member ring with a double bond is given small molecules (e.g., H2O, CO, NH3) are special types of charge remote fragmentation, where the in the first place, there is no need for the preformation of such structures. These three reactions along cleavage is unrelated to the location of charge. The2 retro-Diels-Alder3 reaction (rDA) is also known with the elimination of small molecules (e.g., H2O, CO, NH3) are special types of charge remote from chemicalfragmentation, synthesis. where As the opposed cleavage to is OE,unrelated in the to case the oflocation EE ions, of charge. the process The retro-Diels-Alder is most probably not radical initiated,reaction (rDA) and is involves also known electron from chemical pair migrations synthesis. As in opposed the ring. to OE, This in the type case of of fragmentationEE ions, the can be used e.g.,process for is themost structure probably not elucidation radical initiated, of flavonoids and involves [39 electron,40]. A pair typical migrations rDA in reaction the ring. isThis shown for type of fragmentation can be used e.g., for the structure elucidation of flavonoids [39,40]. A typical theophyllinetype of (a fragmentation xanthine) in can Figure be used5. The e.g., rulesfor the introduced structure elucidation above are of flavonoids successfully [39,40]. applied A typical to identify rDA reaction is shown for theophylline (a xanthine) in Figure 5. The rules introduced above are the possiblerDA fragmentsreaction is shown of moclobemide for theophylline shown (a xanthine in Figure) in 6Figure. 5. The rules introduced above are successfully applied to identify the possible fragments of moclobemide shown in Figure 6.

Figure 5.FigureRepresentation 5. Representation of retro-Diels-Alderof retro-Diels-Alder reaction (rDA), (rDA), a typical a typical charge charge remote remote process processon on protonatedprotonated theophylline. theophylline. The neutral The neutral molecule molecule eliminated eliminated here here isa is characteristic a characteristic loss loss for for other other xanthines as well. Thexanthines name as charge well. The remote name reflects charge thatremote the refl reactionects that takes the reaction place without takes place the involvementwithout the of the involvement of the ionizing proton. Therefore, the exact site of ionizing proton is usually not depicted. ionizinginvolvement proton. Therefore, of the ionizing the exactproton. site Therefore, of ionizing the exact proton site of isionizi usuallyng proton not is depicted. usually not depicted.

Figure 6. The application of all rules concerning fragmentation on the example of protonated moclobemide, a reversible monoamine oxidase inhibitor. IC stands for inductive cleavages and all the fragments could be explained by this route alone. There is no sign of violation of the even-electron rule. Note the characteristic isotope pattern which could be attributed to a single chlorine atom in the precursor and some of the fragments. An even m/z value (rounded to integer) implies an uneven number of in the given ion and vice versa for an even-electron ion which is in concordance with the nitrogen rule. Molecules 2019, 24, 611 9 of 11

5. Software-Aided Structural Analyses The above-mentioned selected examples could be very useful for novices to understand the basics of ESI-MS spectra evaluation. When analyzing compounds possessing multiple functional groups and/or condensed ring systems however, evaluation of MS/MS spectra could be very complicated and interpretation of these spectra is challenging even for experts (see Figure4). In these cases, software-based spectra predictors or compound identifiers serve as a good alternative. A user-friendly and free to download software with free source code for these purposes could be found for example on the website http://cfmid.wishartlab.com/[ 41]. These work by algorithms based on experimental MS/MS spectral databases. Even in these cases, proposed fragment ion structures could not always be easily elucidated. It is also very often for product ion predictors to fail to explain a significant number of observed peaks in the experimental spectrum, especially for compounds that have not been described previously.

6. Summary Here, we made an attempt to bring the field of electrospray-tandem mass spectrometry closer to organic unexperienced in this field. Key pillars of the evaluation of tandem mass spectra of organic compounds can be summarized in the following points:

• selection of ionization mode is based on the basicity of the compound of interest • analytes are ionised to yield even-electron ions in most cases • the fragmentation processes of even-electron ions are much closer to the concepts laid down in organic chemistry as opposed to the ones observed in electron ionization • the resulting fragments are generally even-electron ions • elimination of small neutral molecules from precursors are preferable • most fragment ions are a result of inductive cleavages, acyclic hydrogen rearrangements as well as some (a)cyclic special rearrangements • the identity of a previously described compound in an unknown sample could be determined by using (U)HPLC-ESI-MS/MS and software-based annotation

7. Notes All the MS(/MS) spectra are the own work of the authors and were obtained on an Esquire 3000+ ion trap instrument (Bruker, Bremen, Germany). Pure samples were dissolved in acetonitrile-water (1:1, v/v) mixture containing 0.1% unless otherwise stated.

Funding: This work was completed in the ELTE Institutional Excellence Program (783-3/2018/FEKUTSRAT) supported by the Hungarian Ministry of Human Capacities. Conflicts of Interest: The authors declare no conflict of interest.


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