molecules

Review
An Organic Chemist’s Guide to Electrospray Mass
Spectrometric Structure Elucidation
Arnold Steckel 1 and Gitta Schlosser 2, *
1 Hevesy György PhD School of Chemistry, ELTE Eötvös Loránd University, Pázmány Péter sétány 1/A,
1117 Budapest, Hungary; starnold@caesar.elte.hu
2 Department of Analytical Chemistry, ELTE Eötvös Loránd University, Pázmány Péter sétány 1/A,
1117 Budapest, Hungary
* Correspondence: schlosser@caesar.elte.hu




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


Abstract: Tandem mass spectrometry is an important tool for structure elucidation of natural and
synthetic organic products. Fragmentation of odd electron ions (OE+ ) generated by electron ionization
(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, electrospray ionization (ESI) and atmospheric pressure chemical
ionization (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: tandem mass spectrometry; MS/MS fragmentation; collision-induced dissociation; CID;

ESI; structure elucidation

1. Introduction
Electron ionization (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
electrons with ~70 eV energy. This energy is high enough to yield very reproducible mass spectra with
a large number of fragments. However, these spectra frequently lack the radical type molecular ions
(M+ ) due to the high internal energy 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 biomolecules as well as their
detailed structures could be determined, even when high pressure liquid chromatography 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 Ion for Fragmentation

ESI methodology can be used in either positive (ES+) and negative ion mode (ES−), depending on
the proton affinity of the compound. In ES+, only analytes with at least one positive elementary charge,
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 methodology
basic can be could
characteristics used inbe either
easilypositive
ionised (ES+)inand
ES+ negative ion mode
via making (ES−), depending
adducts with proton(s),
on the proton affinity of the compound. In ES+, only analytes with at least one positive elementary
while molecules having acidic functional groups—and lacking basic ones—typically give 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 acid-base theory [9].
Molecules possessing basic characteristics could be easily ionised in ES+ via making adducts with
It should be emphasized
proton(s), that only
while molecules having electrospray-compatible
acidic functional groups—and solvents (e.g.,basic
lacking acetonitrile, water, give
ones—typically methanol,
ethanol)—containing
better spectra in ES−. Optimal ionization mode selection can be based on the Brønsted-Lowry acid-can be
0.1% formic or acetic acid to enhance protonation—or solvent mixtures
used for
basedissolving samples
theory [9]. It should be during sample
emphasized thatpreparation. Solvents are highly
only electrospray-compatible solventsrecommended to be of
(e.g., acetonitrile,
LC-MSwater,
grade. methanol,
Doublyethanol)—containing
distilled water should 0.1%be formic
usedorfor acetic acid tosolutions
aqueous enhance protonation—or solvent the
to effectively minimize
mixtures
intensity of sodium can be used for
adducts in thedissolving
spectra.samples
It is alsoduring sample
advisable preparation.
to use low-binding,Solvents are highlyplastic
high-quality
recommended to be of LC-MS grade. Doubly distilled water should be used for aqueous solutions to
sample tubes to decrease the otherwise always present plasticizer contamination.
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
ES+, while singly deprotonated [M − H]− ions are dominant in ES−. Multiply protonated
ions incontamination.
species areThe usually
type offormed
EE ionsfrom
generatedlargeinmolecules
the source are (e.g., polymers),
usually singly orormultiply
ones having multiple
protonated favorable
[M + zH] z+

sites for protonation. These ions are fragmented more easily due
ions in ES+, while singly deprotonated [M − H] ions are dominant in ES−. Multiply protonated
− to charge repulsion; however,
species
fragments areare usually
often notformed from large molecules
too informative. If the molecule(e.g., polymers), or ones having
is large enough, it willmultiple favorable by an
be represented
sites for protonation.
envelope-shaped distributionThese of ions
peaks. are fragmented more easily due to charge repulsion; however,
fragments
Charge are often notcan
distributions too informative.
be altered by If the molecule
using is large
buffers. enough,
Adduct it will be represented
formation could also by takean place
envelope-shaped distribution of peaks.
depending on the Lewis basicity of the compound in the presence of NH4 , Na and K or if Li+ + + +
Charge distributions can be altered by using buffers. Adduct formation could also take place
or Ag+depending
is intentionally added to the sample [10,11]. Note that these ions should be referred to as
on the Lewis basicity of the compound in the presence of NH4+, Na+ and K+ or if Li+ or Ag+
cationized moleculesadded
is intentionally as thetoterms
the samplequasi-molecular
[10,11]. Note that ion and
thesepseudo-molecular
ions should be referred ion to
areasdiscouraged
cationized [2].
These molecules
adduct ions usually
as the give no fragmentation
terms quasi-molecular other than the loss
ion and pseudo-molecular of the
ion are cation, but
discouraged for special
[2]. These
compounds
adduct (e.g., carbohydrates),
ions usually their fragmentation
give no fragmentation other than thecould loss ofbethealso significantly
cation, different
but for special compoundsfrom that
+
of [M + H] ions, giving useful structural information [12]. Ammonium acetate or of
(e.g., carbohydrates), their fragmentation could be also significantly different from that [M + H]+ are
bicarbonate
usuallyions,
used giving
for useful
adduct structural
formation information
typically [12].
in Ammonium
5 mM concentrationacetate or bicarbonate
(Figure 1).areItusually
is worth usednoting
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 chloride, trifluoroacetate)
suppression of signal intensity (e.g., phosphate buffers, sodium chloride, trifluoroacetate) should be
shouldavoided.
be avoided.In ES−, ES−, adduct
Inadduct formation formation
could also could
happen; also anionized
happen; anionized
molecules may molecules
be usefulmay in be
theuseful
in the detection
detectionofofsome some compounds
compounds (e.g.,(e.g., carbohydrates,
carbohydrates, flavonoids).
flavonoids).

1. Single
FigureFigure stagestage
1. Single mass spectrum
mass ofoftrehalose,
spectrum trehalose, aa disaccharide found
disaccharide found e.g.,
e.g., in insects.
in insects. Due Due
to theto the lack
lack
of basic
of residues in the
basic residues molecule,
in the molecule,[M
[M++H]H]++ ions
ionscould
could notnot
be be detected
detected if theifsample
the sample was dissolved
was dissolved in
acetonitrile-water1:1
in acetonitrile-water 1:1(v/v)
(v/v) solvent
solvent mixture,
mixture,containing
containing 0.1% acetic
0.1% acidacid
acetic (spectrum not shown).
(spectrum 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 ammonia (*). 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.
 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
are very likely the [M + NH4]+ ion of dibutyl phtalate and [M + H]+ ion of erucamide, respectively,3 of 11
Molecules 2019, 24, 611
which are common plasticizers leaked from plastic sample tubes.

If the
the analyte
analyte isis prone
proneto tocomplex
complexformation
formationororisishighly
highlyconcentrated,
concentrated, [2M
[2M+ H] + [2M
+ or
+ H] or [2M − H]−−
H]−
ions couldcould
ions also bealsopresent in the
be present in full
the mass spectra.
full mass It is It
spectra. also common
is also common to detect M ions
to detect+ +
M ionsusingusingsoft
ionization methods if the compound already had a fixed charge before ionization
soft ionization methods if the compound already had a fixed charge before ionization (Figure 2). (Figure 2). Typical
examples are quaternary
Typical examples ammonium
are quaternary salts. Thesalts.
ammonium distribution of charge of
The distribution viacharge
conjugation also enhances
via conjugation also
the stability
enhances theofstability
the protonated/deprotonated ion as e.g.,
of the protonated/deprotonated inas
ion case ofin
e.g., compounds with a dienone
case of compounds moiety.
with a dienone
If a given
moiety. If acompound
given compound (e.g., ketones, aldehydes,
(e.g., ketones, alcohols)
aldehydes, cannot
alcohols) be detected
cannot in either
be detected mode
in either modeor
fragmentation
or fragmentation efficiency
efficiency needs
needs to to
bebeenhanced,
enhanced,one onecould
coulduse use derivatization
derivatization agents
agents [13]. These
compounds are are usually
usually basic
basic to
toenhance
enhanceionization
ionizationefficiency.
efficiency.The
Thelimitation
limitationofofthis
thisapproach
approach is is that
that it
it cannot
cannot bebeused
usedfor fortotally
totallyunknown
unknowncompounds.
compounds.Another
Anotherway way isis to
to use
use aa different
different ionization type
such as APCI
APCI [14][14]or oratmospheric
atmosphericpressure
pressurephotoionization
photoionization (APPI),
(APPI), which
which maymaybe be
moremore applicable
applicable for
for less polar compounds (e.g., lipids) [15]. Note that molecular +
ions (M +) could be also observed
less polar compounds (e.g., lipids) [15]. Note that molecular ions (M ) could be also observed using
using or
APCI APCIAPPIor [14–16].
APPI [14–16].

Figure 2.
Figure IllustrationofofField’s
2. Illustration Field’srule
ruleononthiamine
thiamine(vitamin
(vitaminB1B ) in
) 1in positive
positive mode
mode (ES+).
(ES+). Note
Note thatthat
the
the precursor in blue is not a protonated thiamine molecule [M + H] + , but simply an M+ , as the
precursor in blue is not a protonated thiamine molecule [M + H] , but simply an M , as the compound
+ +
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 Scheme 1), while the one in green indicates an acyclic hydrogen 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) resonance 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 ion trap 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. Representation

Representationofof thethe
main noncyclic
main fragmentation
noncyclic pathways
fragmentation characteristic
pathways to even-electron
characteristic to even-
ion fragmentation
electron in ES+ mode.
ion fragmentation in ES+Green
mode.arrows
Green indicate inductive
arrows indicate cleavages,
inductive while blue
cleavages, whileones
bluedenote
ones
acyclic β-H-rearrangements. The molecule of interest must contain at least one functional
denote acyclic β-H-rearrangements. The molecule of interest must contain at least one functional group prone
to be protonated.
group prone to beXprotonated.
denotes -NH, -O- or -S-
X denotes groups,
-NH, -O- orwhile Y denotes
-S- groups, while-CH -, C=O, -SO
Y2denotes -CH22-,ones
C=O, so -SO
that2
the
onesgeneral
so thatfragmentation routes for amines,
the general fragmentation routes (sulfon)amides, ethers, esters,
for amines, (sulfon)amides, thioesters
ethers, esters,are given here.
thioesters are
Molecules
given here.featuring
Moleculesgroups not groups
featuring coverednot
bycovered
the scheme above,
by the schemelikeabove,
alcohols
likeoralcohols
carboxylic acids are
or carboxylic
prone to lose
acids are water
prone to or carbon
lose waterdioxide/formic acid upon collision-induced
or carbon dioxide/formic dissociation respectively.
acid upon collision-induced dissociation
Ketones usually dissociate to yield acylium ions, while simple aldehydes
respectively. Ketones usually dissociate to yield acylium ions, while simple are often cannot beare
aldehydes detected
often
without
cannot be derivatization.
detected withoutExpressive and more Expressive
derivatization. specific examples are collected
and more by Niessen
specific examples areet collected
al. [2] (p. 84;
by
or 94–101etfor
Niessen al. general routes
[2] (p. 84; of fragmentation
or 94–101 for specific
for general routes compound classes).
of fragmentation for specific compound classes).

3. Performing MS/MS Experiments

Fragmentation of even-electron ions usually follow the so-called even-electron rule [36,37].
Based Toonobtain structural information
the even-electron from anofEE
rule, the formation ion,
ions withanno MS/MS
unpaired(tandem mass
electrons arespectrometric)
favored upon
experiment
fragmentation, is needed
along witheitherloss
in aof collision
neutralcell or in an from
molecules instrument featuring instead
the precursor, an ion trap analyzer.
of homolytic
Protonated
cleavages whichinitial molecules selected for
are rare amongst thisfragmentation
type of ions.are called precursor
Eliminations ions, while
of neutral their fragments
molecules could be
are referred to(selective
characteristic as product or ions. Theto
specific) terms mother
certain typesand daughter ions
of compounds. Forare
a discouraged
very thorough [2],and
however,
useful
list on characteristic neutral losses, see Niessen et al., 2017 [2].
they are still widely used. Both in traps and collision cells, the analyte ions generated by the ion
sourceHeterolytic
are excited cleavage is oftenwith
by collisions referred
inertto as inductive
gases e.g., N2 , cleavage and can be observed
Ar or He—depending on the for both OE
instrument
and EE ions.
type—to impartThe inductive
enough energycleavage
inducing of the carbon-heteroatom
decomposition processes bond
[17]. isIt ashould
CMF alsoleading to anthat
be noted EE
fragment. Typical
fragmentation inductive
in the cleavage
ion source couldproducts
occur withare represented
considerableinextent
Figurefor3 for protonated
fragile compounds bisoprolol.
or by
A competitive
altering fragmentation
source parameters. Thispathway with inductive
could be exploited in case ofcleavage of EE ionsionistraps
using quadrupole the where
acyclicuseful
β-H-
rearrangement
fragments could(abeCRF) and in
missing could be deducted
standard MS/MSfrom an due
spectra inductive cleavagelimitation
to a technical where a called
protonlow-mass
at the β-
carbon migrates
cut-off. Another to way thefor
other fragment. of
the induction Thus, in softfragmentation
in-source ionization, heterolytic
where there cleavage also include
is no second mass
acyclic β-H-rearrangement.
analyser (e.g., single quadrupole For both pathways,
instruments) the sum ofcone
is performing the two fragments
voltage is equal to M + 2H+,
fragmentation/declustering
and the relative
potential abundance
fragmentation. of them
These methodsis determined
could onlybybethe Field’s
used rule. Inductive
however cleavage
if the compound is and acyclic
purified or
β-H-rearrangement
MS is hyphenated with are two of the
HPLC. most important
Investigation of thecleavage types in processes
fragmentation the dissociation
in caseofofEE EEions.
+ ions are
usually carried out using various methods, such as theoretical calculations, stable isotope labelling,
H/D exchange [18] and MSn experiments on instruments capable of high resolution and high mass
accuracy (HRAM-MS/MS). Orbitrap 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 masses 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 energies imparted to precursors) or is coupled to ion
Molecules 2019, 24, 611 5 of 11

mobility spectrometry, discrimination between constitutional isomers 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 protons 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 peptides [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 (Table 1).

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]−
Nitrogen rule 3 3
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 laser desorption/ionization, APCI: atmospheric pressure
chemical ionization, APPI: atmospheric pressure photoionization. 2 RDBE: ring and double bond equivalent. RDBE
should be calculated as NX − N2Y + N2Z + 1 where X = C, Si; Y = H or halogens, Z = N, P ([1], p. 28, [28]) Note
that sulfur and oxygens atoms 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 electron transfer dissociation (ETD) [30] which
nowadays have widely-used application in sequencing 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 Figure 2.
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 bromine, chlorine, certain transition metals or intentionally introduced stable isotopes 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] (Table 2): 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 atom 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 Scheme 1. For more specific examples, see Figures 3–6 (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 Figure 3 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
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Molecules 2019, 24, x FOR PEER REVIEW 7 of 11

3. Tandem
FigureFigure 3. Tandem massmassspectrum
spectrumofofbisoprolol,
bisoprolol, a cardioselective The The
β-blocker.
cardioselective β-blocker. initialinitial
site ofsite of
protonation is
protonation the basic
is the basicspsp33 N.
N. Without
Without thisthis moiety,
moiety, the molecule
the molecule cannot becannot
detected.be detected.
After collision- After
Figure 3. Tandem mass spectrum of bisoprolol, a cardioselective β-blocker. The initial site of
induced activation, the precursor ion [M +[M
H]++ can+ be represented by two structures. All two
collision-induced
protonation isactivation,
the basic spthe
3 N.precursor
Without this ionmoiety, H] can be represented
the molecule by twoAfter
cannot be detected. structures.
collision-All two
fragments
fragments of the of the precursor
precursor ion are a result of inductive cleavages in concordance with Scheme 1, green
induced activation, theion are a result
precursor of inductive
ion [M + H] can cleavages
+ in concordance
be represented with Scheme
by two structures. 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, X = O, Yion
the precursor = CH
are 2a).result
Note ofthat thesecleavages
inductive fragments not necessarily
in concordance represent
with Scheme the most
1, green
stable resonance structures.
stable arrows (in this
resonance case, X = O, Y = CH2). Note that these fragments not necessarily represent the most
structures.
stable resonance structures.
Homolytic cleavages include σ- (mostly for OE) and α-cleavage (only for OE). The role of σ-bond
Homolytic
homolytic
cleavages
cleavage
include
in EE+include
σ- (mostly
fragmentation
for OE)are
processes
and α-cleavage (only for OE). The role of σ-bond
Homolytic cleavages σ- (mostly for OE) andlimited to certain
α-cleavage (onlytypes of compounds
for OE). having
The role of σ-bond
homolytic cleavage in
a carbon-heteroatom EE+ fragmentation processes are limited to certain types of compounds having
homolytic cleavage in bond resulting in the
EE+ fragmentation loss ofare
processes radical species
limited suchtypes
to certain as Cl˙,
· of NO 2˙, ˙CH3, CH3O˙
compounds
· · having·
a carbon-heteroatom bond resultingof in the loss of rule,
radical species4.such as Cl , NO 2 2,˙,CH 3 ,3,CH O [3,27].
a[3,27]. For this type
carbon-heteroatom of bond
violation even-electron
resulting see Figure
in the loss of radical speciesThese
suchlosses ofNO
as Cl˙, radicals are
˙CH usually
CH33O˙
For this type of
competitive violation
with the of
loss even-electron
of their rule,
non-radical see Figure
counterparts 4.
e.g., These
HCl. losses of radicals
[3,27]. For this type of violation of even-electron rule, see Figure 4. These losses of radicals are usually are usually
competitive with the loss of their non-radical counterparts
competitive with the loss of their non-radical counterparts e.g., HCl.e.g., HCl.

Figure 4. In-source fragmentation of protonated tianeptine, marketed as a nootropic ‘dietary

supplement’
4. In-source in thefragmentation
USA. Note that m/z 213 and 193 correspond to fragments that violate the above-
FigureFigure 4. In-source fragmentation ofof protonated tianeptine,
protonated tianeptine, marketed
marketed as aasnootropic
a nootropic ‘dietary
‘dietary
mentioned even-electron rule. Elimination of SO2 is a charge-remote neutral loss characteristic to
supplement’ in the
supplement’ USA.
in the USA.Note
Note that m/z
that m/z 213213
and and 193 correspond
193 correspond to fragments
to fragments that violatethat violate the
the above-
sulfonamides [38], sulfonic acids and aromatic sulfoxides. The mechanisms for losses and
mentioned even-electron
above-mentioned even-electronrule. Elimination
rule. Elimination 2 is
of SOof SO a charge-remote neutralneutral
is a charge-remote loss characteristic to
loss characteristic
rearrangement reactions are often not trivial but rules2 known from organic chemistry regarding
sulfonamides[38],
to sulfonamides [38], sulfonic
sulfonic acids
acids and
and aromatic
aromatic sulfoxides.
sulfoxides.The The
mechanisms
mechanismsfor losses
for and and
losses
stability of ions could be successfully applied.
rearrangement reactions are often not trivial but rules known from organic chemistry regarding
rearrangement reactions are often not trivial but rules known from organic chemistry 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,
Molecules 2019, 24,
24, xx FOR
FOR PEER
PEER REVIEW
REVIEW 88 of
of 11
11

It is often required for atoms and bonds to be rearranged either through a six-membered cyclic
It is often required for atoms and bonds to be rearranged either through a six-membered cyclic
reaction.transition
Didactic examples
state or
for these
or through
through ion/neutral
types are
ion/neutral complexes
reviewed
complexes prior
by Demarque
prior to to fragmentation.
et al. [6].
fragmentation. Most
A common
retro-heteroene
Most common
transition state
reaction could be considered
rearrangement types
rearrangement types for as the
for protonatedeven-electron
protonated molecules counterpart
molecules include
include retro of McLafferty
retro (hetero)ene
(hetero)ene reactions rearrangement.
reactions and
and retro Diels-In case of
retro Diels-
retro-Diels-Alder
Alder reaction,
Alder reaction.
reaction. theexamples
Didactic
Didactic six-member
examples ringtypes
for these
for these withare
types a double
are reviewed
reviewed bond is given in
by Demarque
by Demarque et the
et first
al. [6].
al. [6]. AA place,
retro- there is
retro-
heteroene
no need heteroene reaction could
for the preformation
reaction could be considered
of such
be considered
structures.as asThese
the even-electron
the even-electron
three reactions counterpart
along with
counterpart of the
of McLafferty
elimination of
McLafferty
rearrangement. In
rearrangement. In case ofof retro-Diels-Alder
retro-Diels-Alder reaction,
reaction, thethe six-member
six-member ring ring with
with aa double
double bond
bond is is given
given
small molecules (e.g., Hcase2 O, 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
cleavageinisthe
with
first place, there is no need for the preformation of such structures. These three reactions along
unrelated
the eliminationto theoflocation
elimination of charge.
small molecules
molecules (e.g., The O, retro-Diels-Alder
H22O, CO, NH NH33)) are specialreaction
are special types ofof (rDA) is also known
charge remote
remote
with the of small (e.g., H CO, types charge
from chemical synthesis.
fragmentation, where
fragmentation,
As
where the opposed
the cleavage
cleavage isto OE, in
is unrelated the
unrelated to case
to the of EE
the location ions,
location of the
of charge. process
charge. The is most
The retro-Diels-Alder
retro-Diels-Alderprobably not
radical initiated,
reaction
reaction (rDA)andisinvolves
(rDA) is also known
also known electron
from pair migrations
chemical
from chemical synthesis. Asin
synthesis. As the ring.
opposed
opposed to OE,
to OE, This
in type
the
in the caseof
case of fragmentation
of EE ions, the
EE ions, the can
process
be used e.g., is most
foristhe
process probably
moststructure
probably not not radical
elucidation initiated, and
of flavonoids
radical initiated, involves
and involves electron
[39,40]. pair
electronApair migrations
typical rDA in
migrations in the
reactionring.
the ring.is This
shown for
This
type of
type of fragmentation
fragmentation can be be used
used e.g.,
e.g., for the
the structure
structure elucidation
elucidation of of flavonoids [39,40].
[39,40]. A A typical
typical
theophylline (a xanthine) incan Figure 5. The for rules introduced above areflavonoids
successfully applied to identify
rDA reaction
rDA reaction is is shown
shown for for theophylline
theophylline (a (a xanthine)
xanthine) in in Figure
Figure 5. 5. The
The rules
rules introduced
introduced above above areare
the possible fragments
successfully appliedof moclobemide
to identify the possibleshownfragments
in Figure 6.
of moclobemide shown in Figure 6.
successfully applied to identify the possible fragments of moclobemide shown in Figure 6.

Representation
Figure 5.Figure
Figure 5. of retro-Diels-Alder
5. Representation
Representation retro-Diels-Alder reaction
of retro-Diels-Alder
of (rDA),
reaction (rDA),
reaction (rDA), a typical
aa typical
typical charge
charge
charge remoteremote
remote process process
process on
on on
protonated
protonated theophylline.
theophylline. The The
neutralneutral molecule
molecule eliminated
eliminated here here
is a is a characteristic
characteristic loss
loss
protonated theophylline. The neutral molecule eliminated here is a characteristic loss for other forfor other
other xanthines
xanthines
as well. The ascharge
nameas
xanthines well. The
well. The namereflects
remote
name charge that
charge remote reflects
thereflects
remote reaction that
that the reaction
takes
the reaction takes place
place without
takes place without the
the of the
the involvement
without
involvement of
involvement of the
the ionizing
ionizing proton.
proton. Therefore,
Therefore, the
the exact
exact site
site of
of ionizing
ionizing proton
proton is
is usually
usually not
not depicted.
depicted.
ionizing proton. Therefore, the exact site of ionizing proton is 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 nitrogens 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 Figure 4). 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 chemists 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% acetic acid 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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