A Unique Collision-Induced Dissociation Reaction of Cholamine Derivatives of Certain Prostaglandins

Article abstract of DOI:10.1007/s13361-018-2051-6

Prostaglandins (PGs) are biologically active metabolites of arachidonic acid containing 20 carbon atoms, a cyclic moiety, and two side chains (A and B) in common. The bioassay of PGs requires high sensitivity because of their low concentration in tissues and blood and has usually been carried out by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the negative ion mode. Chemical derivatization of PG carboxylic acid groups to introduce positive charge-carrying groups is an established strategy to improve the sensitivity and selectivity of such assays. In this study, we exploited this approach for structural identification of a series of PGs using cholamine derivatization through an amidation reaction. However, we observed that collision-induced dissociation of these derivatives gave rise to unexpected product ions that we postulated were formed by unique long-range intramolecular reactions resulting in dehydration of the B chain accompanied by fragmentation of the A chain through an unusual Hofmann rearrangement. Evidence for the proposed mechanism is presented based on ESI-MS/MS and high resolution mass spectrometry studies of cholamine derivatives of PGE₁, PGE₂, PGD₂, PGI₂, and C-17 methyl deuterium-labeled limaprost. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s13361-018-2051-6

B American Society for Mass Spectrometry, 2018
J. Am. Soc. Mass Spectrom. (2018)
DOI: 10.1007/s13361-018-2051-6
RESEARCH ARTICLE
A Unique Collision-Induced Dissociation
Reaction of Cholamine Derivatives of Certain Prostaglandins
Dong Sun,1,2 Chong Su,1 Yingze Liu,1 Xiangjun Meng,1 John Paul Fawcett,3 Yingjie Guo,1
Jingkai Gu1
1Research Center for Drug Metabolism, School of Life Sciences, Jilin University, Changchun, 130012, People’s Republic of China
2Beijing Xiuzheng Innovation Medicine Research Institute Co., Ltd, Beijing, 102209, People’s Republic of China
3School of Pharmacy, University of Otago, P.O. Box 56, Dunedin, New Zealand
Abstract. Prostaglandins (PGs) are biologically
active metabolites of arachidonic acid containing
20 carbon atoms, a cyclic moiety, and two side
chains (A and B) in common. The bioassay of
PGs requires high sensitivity because of their low
concentration in tissues and blood and has usually
been carried out by electrospray ionization tandem
mass spectrometry (ESI-MS/MS) in the negative
ion mode. Chemical derivatization of PG carboxylic
acid groups to introduce positive charge-carrying
groups is an established strategy to improve the sensitivity and selectivity of such assays. In this study, we exploited
this approach for structural identification of a series of PGs using cholamine derivatization through an amidation
reaction. However, we observed that collision-induced dissociation of these derivatives gave rise to unexpected
product ions that we postulated were formed by unique long-range intramolecular reactions resulting in dehydration
of the B chain accompanied by fragmentation of the A chain through an unusual Hofmann rearrangement. Evidence
for the proposed mechanism is presented based on ESI-MS/MS and high resolution mass spectrometry studies of
cholamine derivatives of PGE1, PGE2, PGD2, PGI2, and C-17 methyl deuterium-labeled limaprost.
Keywords: Electrospray ionization tandem mass spectrometry, Prostaglandins, Cholamine derivatives, Macrocyclic
intermediate, Hofmann rearrangement
Received: 27 April 2018/Revised: 18 July 2018/Accepted: 4 August 2018
tion to introduce positive charge-carrying groups has been
applied to impart high-ionization efficiency for the lipid acid
Introduction
rostaglandins (PGs) are important participants in the regu-
lation of physiological activity in various tissues of humans
analytes in the positive ion mode [1–5].
P
Various quaternary ammonium derivatization reagents have
been applied to introduce such moieties to PG molecules such
as salts of the N-(4-aminomethylphenyl)pyridinium (AMPP)
and (2-(4-aminophenoxy)ethyl) (4-bromophenethyl)dimethyl
ammonium (4-APEBA) ions [6–11]. Among them, cholamine
yields benefits not only with regard to the improved signal
response of precursor ions but also to their greater stability
due to the formation of a dihydrooxazolium ring [10]. Further-
more, these stabilized product ions are derived from the analyte
residue rather than from the derivatization reagent which en-
ables further structure-related fragmentation studies.
and animals. All PGs are synthesized in the cell from the
essential fatty acid, arachidonic acid. Structurally, they contain
20 carbon atoms with at least one ring structure, one 7-carbon
A chain with a terminal carboxylic acid group and one 8-
carbon B chain incorporating an allylic alcohol moiety. Bioas-
say of PGs by liquid chromatography tandem mass spectrom-
etry (LC-MS/MS) in the negative ion mode usually requires an
acidic mobile phase which tends to decrease the ionization
efficiency of the carboxylic acid group. Therefore, derivatiza-
Prostaglandin E1 (PGE1, also known as alprostadil) has been
clinically available for many years as an injectable vasodilator
Correspondence to: Yingjie Guo; e-mail: guoyingjie@jlu.edu.cn, Jingkai Gu;
e-mail: gujk@jlu.edu.cn

D. Sun et al.: A CID Reaction of Cholamine Derivatives of Prostaglandins
and inhibitor of platelet aggregation. A synthetic analog of PGE1,
1 mM 1-[bis(dimethylamino)methylene] -1H-1,2,3-
triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate
(HATU) in acetonitrile were added and the mixture incubated
for 15 min at ambient temperature. Then a 100 μL aliquot of an
aqueous solution of 1 mM cholamine or ethanolamine was
added and the mixture incubated for 1 h at ambient tempera-
ture. Previous research showed that an incubation of 1 h dura-
tion is sufficient for the reactions to go to completion.
limaprost, is more stable than PGE1 and can be taken orally. In
the collision-induced dissociation (CID) of cholamine-
derivatized limaprost (1), a series of intense signals arising from
exclusive neutral mass loss was observed [10] together with a set
of weak but highly specific product ions in the fragmentation
pattern of 1. These additional product ions are also present in the
MS2 and MS3 spectra of cholamine derivatives of other structur-
ally similar prostaglandins viz PGE1, PGE2, and PGD2. Such
fragmentation behavior is reminiscent of the structurally specific
McLafferty rearrangement which is observed in the MS of mol-
ecules containing a keto-group undergoing β-cleavage.
In the present study, we used electrospray ionization tandem
mass spectrometry (ESI-MS/MS) and high-resolution time-of-
flight mass spectrometry (HR-TOFMS) to investigate the mech-
anism of formation of these additional product ions in the CID
fragmentation patterns of cholamine-derivatized prostaglandins.
We maintain an understanding of this common fragmentation
behavior will be helpful in future MS-based lipidomic studies.
Mass Spectrometry
MS was performed on a triple TOF 5600 mass spectrometer
(SCIEX, Concord, Canada) and a QTRAP 5500 mass spec-
trometer (SCIEX, Toronto, Canada), each equipped with a
TurboIonSpray™ ESI source. Data acquisition was achieved
using Analyst software 1.6.1 and 1.5.2. In fragmentation ex-
periments, analyte standard solutions were infused into the MS
through a syringe pump at 10 μL/min. Optimized MS param-
eters were as follows: Source temperature 0 °C; ion spray
voltage 5500 V; nebulizer, heater, and curtain gas (N2) 20, 0,
20 psi respectively. DP, CE, and AF2 values were tuned
manually in each experiment.
Materials and Methods
Derivatization Protocol
Results and Discussion
Fragmentation of Cholamine-Derivatized
Limaprost
All chemicals were of analytical grade and purchased
from Sigma (St. Louis, MO, USA). The derivatization
protocol was as follows: To an aliquot (1 mL) of a
3 μM prostaglandin solution in acetonitrile, 100 μL ali-
quots of 1 mM N,N-diisopropylethylamine (DIPEA) and
In our previous research, to identify product ions of cholamine-
derivatized limaprost (1), a set of stabilized positive CID
Figure 1. Product ion scan of cholamine-derivatized limaprost (1) from m/z 100 to 500 collected by QTRAP 5500 MS with a
TurboIonSpray™ ESI

D. Sun et al.: A CID Reaction of Cholamine Derivatives of Prostaglandins
product ions of the parent ion at m/z 465.5 were observed
(Figure 1) [10]. The set included ions at m/z 406.4 (1a, 1-
NMe3), 388.3 (1b, 1-NMe3-H2O), and 370.3 (1c, 1-NMe3-
2H2O) formed by stepwise neutral mass loss. In addition to
the doubly dehydrated product ion 1c, the MS2 spectrum of 1b
also contained a set of specific product ions with weaker
intensity at m/z 360.4 (1d), 344.4 (1e), and 316.4 (1f)
(Scheme 1). These arise from two independent fragmentation
pathways via 1d in one case (shown in blue) and 1e in the other
(shown in red) (Scheme 2). These unique fragmentation path-
ways culminating in 1f therefore involve sequential mass loss
of 28 and 44 Da in one case and 44 and 28 Da in the other.
There was no interchange between the two intermediate frag-
ments and no connection with the dehydration pathway leading
to 1c.
possibility of other fragmentation pathways as found here. A
mechanistic explanation of these alternative pathways based on
the rules of gas phase reactions is shown in Scheme 2.
The first step involves an intramolecular cyclization through
nucleophilic attack of the C-15 hydroxyl group on the
iminoester carbon. Such a long range intramolecular interaction
between the C-15 hydroxyl group of the B chain and the
carboxylate anion of the A chain has been postulated to explain
the specific CID of PGE2 using ESI-MS in the negative ion
mode [12]. Furthermore, formation of a macrocyclic interme-
diate may not be favorable in solution but is possible in the gas
phase or under vacuum as has been found to occur in a CID
reaction of the [MH]+ ions of Leu-enkephalin amide and a
variety of heptapeptide amides [13]. The fact that 1c has the
highest signal intensity among the product ions derived from
1b is due to the high-energy barrier of ester exchange reactions.
In the blue route in Scheme 2, a [1, 3] sigmatropic double-
bond shift initiates a Hofmann rearrangement leading to an
unusual isocyanate that decomposes spontaneously with neu-
tral mass loss of CO (28 Da). Subsequently, an ethylene oxide
(or possibly an acetaldehyde, 44 Da) moiety departs from 1d
leaving the amine product ion 1f.
Mechanism of 1b → 1f Pathways
The elimination of the C-15 hydroxyl group of PGs is known to
involve a [1,3]-sigmatropic rearrangement with a high-energy
barrier to double-bond migration [12]. Therefore, the active
intermediate 1b will tend to accumulate leading to the
Scheme 1. Product ions of cholamine-derivatized limaprost (1)

D. Sun et al.: A CID Reaction of Cholamine Derivatives of Prostaglandins
Scheme 2. Mechanistic explanation of the CID of cholamine-derivatized limaprost
In contrast, in the red route in Scheme 2, the elimination of
an ethylene oxide moiety precedes the Hofmann rearrangement
leading to the same amine product ion 1f. Since 1f is the only
product ion resulting from both pathways and since an amine
group has excellent ionization efficiency, the signal intensity of
1f is expected to be higher than the signals of its precursor ions
1d and 1e. This is actually what is observed (Table 1).
Evidence for the Mechanism
HR-TOFMS and Deuterium Labeling MS2 and MS3 rela-
tionships alone are not adequate to validate a fragmenta-
tion mechanism. Accordingly, the exact mass of all prod-
uct ions derived from 1 were measured by HR-TOFMS
and found to match their corresponding theoretical values
Table 1. HR-TOFMS Data of Cholamine-Derivatized Limaprost (1), C-17 Methyl Deuterium-Labeled Limaprost (2) and PGE1 (3)
Molecule
[M-NMe3-H2O]+
[M-NMe3-2H2O]+
[M-NMe3-H2O-28]+
[M-NMe3-H2O-44]+
[M-NMe3-H2O-72]+
1
Theor. value (/Da)
388.2788 (1b)
388.2846
−15
370.2741 (1c)
370.2696
−12
360.2897 (1d)
360.2855
−12
344.2584 (1e)
344.2533
−15
316.2635 (1f)
316.2596
−12
HRMS (m/z−1
RE δ (/ppm)
)
Relative intensity
Peak CE(/eV)
18.2
5.2
1.3
1.0
3.4
34
41
43
42
43
2
3
Theor. value (/Da)
391.3035
391.3028
−2
362.2690
362.2658
−9
30.5
32
373.2929
373.2942
+3
344.2584
344.2539
−13
7.0
39
363.3085
363.3091
+2
334.2741
334.2695
−14
1.0
43
347.2772
347.2774
+1
318.2428
318.2410
−6
2.7
42
319.2823
319.2819
−1
290.2478
290.2447
−11
8.5
45
HRMS (m/z−1
RE δ (/ppm)
)
Theor. value (/Da)
HRMS (m/z−1
RE δ (/ppm)
)
Relative intensity
Peak CE(/eV)

D. Sun et al.: A CID Reaction of Cholamine Derivatives of Prostaglandins
Scheme 3. CID patterns of cholamine-derivatized limaprost and other structurally similar PGs
with relative errors (RE) of −15 to −12 ppm (Table 1).
Subsequently, the cholamine derivative of C-17 methyl
deuterium-labeled limaprost (2) was studied to eliminate
the possibility of coincidental matches. In this case,
theoretical values were matched with relative errors of only
−2 to +3 ppm (Table 1). HR-TOFMS of PGE1 (3) with
relative errors of −6 to −14 ppm (Table 1) also supports the
proposed mechanism.
Scheme 4. Mechanism of the Hofmann rearrangement

D. Sun et al.: A CID Reaction of Cholamine Derivatives of Prostaglandins
Scheme 5. Alternative mechanism for the fate of the macrocyclic intermediate (1b*)
Other possible explanations for neutral mass loss of 28 Da
such as ethylene or of 44 Da such as carbon dioxide were ruled
out by HR-TOFMS. On the other hand, a study of the frag-
mentation of benzyl isocyanate showed neutral loss of CO and
formation of benzylamine in support of the proposed
mechanism.
that the C-2 double bond of limaprost does not participate in or
influence the CID fragmentation.
CID of Ethanolamine Derivatized PGs The classic Hofmann
rearrangement (Scheme 4) occurs during reaction of a primary
amide with bromine under basic conditions when loss of CO
from an isocyanate intermediate produces a primary amine
[14]. Formation of 1d from the iminoester 1b* can be consid-
ered a Hofmann rearrangement because it proceeds through an
isocyanate intermediate and involves loss of CO but the reac-
tion is unusual in that it occurs under acidic conditions and the
product is a secondary rather than primary amine. Although
loss of CO from 1e produces a primary amine 1f, it can also be
considered an unusual Hofmann rearrangement because it oc-
curs under acidic conditions.
Instead of a Hofmann rearrangement, a noncyclic amide
(1b**) is also a plausible contributor to the signal at m/z 388.3
and a potential intermediate in the subsequent neutral mass loss
reactions (Scheme 5). To examine this possibility, ethanol-
amine derivatized limaprost (6), C-17 methyl deuterium la-
beled limaprost (7), PGE1 (8), and PGE2 (9) were studied
(Figure 2).
Effect of the C-2 Double Bond In the proposed mechanism,
the double bond in the A chain of limaprost does not play a
role. To verify that this is the case, experiments with
cholamine-derivatized PGE1 (3), PGE2 (4), and PGD2 (5) were
carried out. These prostaglandins have the same carbon skele-
ton as limaprost but without the C-2 double bond in the A
chain. The MS2 spectrum of 3 resulting from m/z 439.4 in-
cludes three very intense product ions at m/z 380.3 (-NMe3),
362.3 (-NMe3, -H2O), and 344.4 (-NMe3, -2H2O). The MS3
spectrum of m/z 362.3 then includes three independent path-
ways viz the second dehydration to give m/z 344.4, sequential
loss of 28 then 44 Da to give m/z 334.3 and 290.3, and
sequential loss of 44 then 28 Da to give m/z 318.3 and 290.3.
The MS2 and MS3 spectra of 4 and 5 also show the same
fragmentation pattern as 3 (Scheme 3). On this basis, it is clear
Figure 2. Ethanolamine derivatives of limaprost and other structurally similar PGs

D. Sun et al.: A CID Reaction of Cholamine Derivatives of Prostaglandins
The intermediate 1b** could be one of the CID dehydration
products of 6. In MS2 and MS3 experiments on 6, 7, 8, and 9,
the following common CID behavior was observed: (a) single
dehydration; (b) double dehydration; (c) mass loss of 28 then
44 Da from b; (d) mass loss of 44 then 28 Da from b (Table 2).
When compared with cholamine derivatives, the signals of
product ions arising from ethanolamine derivatives are roughly
one to two orders of magnitude weaker. These results illustrate
that the amide 1b** may not be a popular fragmentation inter-
mediate in this process. The weak intensities are likely to result
from the poorer leaving group ability of a water molecule in the
ethanolamide groups of 6, 7, 8, and 9. By losing one, two, or
three water molecules, the CID pathways of ethanolamine
derivatives may include formation of the stabilized cationic
intermediate 1a, 1b, and 1c respectively.
Leaving Group Effects In order to identify the role of the
leaving group in producing the parallel CID fragmentation
patterns, we prepared an ethylamine derivative of limaprost
(10, Figure 3). No trace of sequenced mass loss of 28/44 and
44/28 Da was observed in the MS2 of 10 consistent with the
absence of a leaving group.
Figure 3. Compounds designed to evaluate the leaving group
effect
We next examined cholamine-derivatized heptanoic
acid (11) in order to determine whether a stabilized
dihydrooxazolium cation structurally similar to the A
chain of cholamine-derivatized PGE1 (3) would undergo
the neutral mass loss sequence. In the CID pattern of 11,
neutral mass loss of trimethylamine (−59 Da) is clearly
evident but there was no signal indicative of sequential
mass loss of 28/44 and 44/28 Da. Therefore, a stabilized
cationic intermediate is not a sufficient condition for
triggering the unique type of Hofmann rearrangement
observed here.
(12, Figure 4). PGI2 differs from other PGs in incorporating an
annealed bicyclic structure. Due to restriction imposed by its
molecular skeleton, particularly the Z-double bond in the A
chain, it is unable to form an intermediate of the form of 1b*.
The MS2 spectrum of 12 derived from m/z 437.4 includes three
intense product ions at m/z 378.3 (-NMe3), 360.4 (-NMe3, -
H2O), and 342.4 (-NMe3, -2H2O) but there was no sequenced
mass loss of 28/44 or 44/28 Da.
In summary, the results in toto support the view that the
unusual fragmentation pattern of cholamine-derivatized
PGs is specifically derived from the macrocyclic interme-
diate 1b*. The imidoester group in 1b* benefits from the
[1, 3] sigmatropic rearrangement in the B chain and
rearranges to the energetically more stable amine. This
cascade rearrangement occurs in parallel with the elimina-
tion of ethylene oxide and gives rise to the unique CID
fragmentation pattern.
The Role of the Macrocyclic Intermediate 1b* Formation of
the intermediate 1b* is crucial in giving rise to the parallel
fragmentation pathways of our postulated mechanism. Evi-
dence for the formation of 1b* is not only provided by struc-
turally similar PGs, but also by cholamine-derivatized PGI2
Table 2. Fragmentation Patterns of Ethanolamine-Derivatized Limaprost (6),
C-17 Methyl Deuterium-Labeled Limaprost (7), PGE1 (8), and PGE2 (9)
(Confirmed via MS3)
Precursor
-H2O
-2H2O
(m/z−1
-2H2O-28/44
(m/z−1
-2H2O-72
(m/z−1
ion (m/z−1
)
(m/z−1
)
)
)
)
6
7
8
9
424.6
427.6
398.2
396.2
406.5
409.5
380.5
378.2
388.5
360.2 (−28, w)
344.2 (−44, w)
363.5 (−28, w)
347.5 (−44, w)
334.2 (−28, w)
318.4 (−44, w)
332.2 (−28, w)
316.2 (−44, w)
316.3 (t)
319.3 (t)
290.3 (t)
288.3 (t)
391.5
362.5
360.3
w weak, t trace
Figure 4. The cholamine derivative of PGI2 (12)

D. Sun et al.: A CID Reaction of Cholamine Derivatives of Prostaglandins
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Conclusion
Specific parallel neutral mass loss pathways of 28/44 and 44/
28 Da occur in the CID of cholamine-derivatized limaprost,
PGE1, PGE2, and PGD2 under ESI-positive ion conditions.
Such sequenced mass loss relates specifically to the molecular
structure of cholamine-derivatized PGs and their ability to form
a stabilized dihydrooxazolium cationic intermediate. This sta-
bilized intermediate facilitates the formation of a unique mac-
rocyclic product ion when a [1, 3] sigmatropic rearrangement
in the B chain is coupled to and initiates an unusual Hofmann
rearrangement in the A chain. At the same time, the elimination
of an ethylene oxide occurs and gives rise to the specific CID
pattern. Such specific fragmentation behavior in the cholamine
derivatives of structurally similar PGs provides useful informa-
tion in the lipidomic profiling of prostaglandins and their
analogs.
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The authors gratefully acknowledge financial support from the
National Natural Science Foundation of China (Grant Nos.
81430087 and 81673396) and the Science and Technology
Major Specialized Projects for Bsignificant new drugs creation^
of the 13th five-year plan (2017ZX09101001).
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