Dehydration-fragmentation mechanism of cathinones and their metabolites in ESI-CID

Article abstract of DOI:10.1002/jms.4538

Various cathinone-derived designer drugs (CATs) have recently appeared on the drug market. This study examined the mechanism for the generation of dehydrated ions for CATs during electrospray ionization collision-induced dissociation (ESI-CID). The genera

Full text of DOI:10.1002/jms.4538

Matsuta Shuntaro (Orcid ID: 0000-0003-4716-4830)
Dehydration-fragmentation mechanism of cathinones and their
metabolites in ESI-CID
Short Tittle: Dehydration-fragmentation mechanism of cathinones in ESI-CID
Shuntaro Matsuta1,2 *, Noriaki Shima , Hidenao Kakehashi , Akari Ishikawa , Ryutaro Asai ,
1
1
1
1
1
1
1
1
2
Atsushi Nitta , Misato Wada , Shihoko Nakano , Hiroe Kamata , Yoshio Nishiyama ,
2
2
1
Hirohisa Nagatani , Hisanori Imura and Munehiro Katagi
1
Forensic Science Laboratory, Osaka Prefectural Police Headquarters, 1-3-18 Hommachi,
Chuo-ku, Osaka 541-0053, Japan.
Division of Material Chemistry, Graduate School of Natural Science and Technology,
Kanazawa University, Kakuma, Kanazawa 920-1192, Japan.
2
*
Mail to: matsutash@gmail.com
Tel: +81-6-6268-1234
Fax: +81-6-6271-8066
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/jms.4538
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Abstract
Various cathinone-derived designer drugs (CATs) have recently appeared on the drug
market. This study examined the mechanism for the generation of dehydrated ions for CATs
during electrospray ionization collision-induced dissociation (ESI-CID). The generation
mechanism of dehydrated ions is dependent on the amine classification in the cathinone
skeleton, which is used in the identification of CATs. The two hydrogen atoms eliminated
during the dehydration of cathinone (primary amine) and methcathinone (secondary amine)
were determined, and the reaction mechanism was elucidated through the deuterium labeling
experiments. The hydrogen atom bonded to the amine nitrogen was eliminated with the
proton added during ESI, in both of the tested compounds. This provided evidence that CATs
with tertiary amine structures (such as dimethylcathinone and α-pyrrolidinophenones
(α-PPs)) do not undergo dehydration. However, it was shown that the two major tertiary
amine metabolites (1-OH and 2″-oxo) of CATs generate dehydrated ions in ESI-CID. The
dehydration mechanisms of the metabolites of α-pyrrolidinobutiophenone (α-PBP) belongs to
α-PPs were also investigated. Stable-isotope labeling showed the dehydration of the 1-OH
metabolite following a simple mechanism where the hydroxy group was eliminated together
with the proton added during ESI. In contrast, the dehydration mechanism of the 2″-oxo
metabolite involved hydrogen atoms in three or more locations along with the carbonyl group
oxygen, indicating that dehydration occurred via multiple mechanisms likely including the
rearrangement reaction of hydrogen atoms. These findings presented herein indicate that the
dehydrated ions in ESI-CID can be used for the structural identification of CATs.
Keywords: cathinones; fragmentation mechanism; ESI-CID-MS/MS; dehydration; designer
drugs
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1
Introduction
Cathinone-derived designer drugs (CATs) are structural analogs of cathinone (Figure 1a),
which is an active component of Khat (evergreen tree leaves). CATs, when consumed, exert a
central nervous system excitatory effect, which is similar to typical stimulants [1–4]. Since
the year 2000, many CATs containing modified chemical structures have been distributed to
evade legal regulations. These changes to the CAT structure usually consist of modifications
to the type of amine group, substituents added on the benzene ring, and alteration to the
length of the alkyl chain. Therefore, it has become essential in the field of forensic science to
identify the slightly modified structures of CATs.
We have previously reported on analytical techniques to identify the structures of CATs in
powder, solution, and biological samples using gas chromatography-electron ionization-mass
spectrometry (GC-EI-MS) and liquid chromatography-electrospray ionization-tandem mass
spectrometry (LC-ESI-MS/MS) [5–10]. In particular, the use of the LC-ESI-MS/MS can
target a wide range of compounds from low to high polarity, and the structures of compounds
can be identified based on the product ion spectra generated in the electrospray ionization
collision-induced dissociation (ESI-CID) following LC separation.
In this study, the generation mechanism of dehydrated product ions in ESI-CID was
examined. Dehydrated ion generation appears to be dependent on the partial structure of the
cathinone skeleton. Dehydrated ions have been observed from both primary and secondary
amine structures such as 3,4-dimethylcathinone, methcathinone, 3,4-dimethylmethcathinone,
N-propylcathinone, hexedrone and ethylone [10–18], while tertiary amine structures such as
4
-chloro-N,N-dimethylcathinone and α-pyrrolidinophenones (α-PPs), do not generate the
dehydration ion [6–9,14–20]. Thus, the dehydrated ions can possibly be used for the
structural identification of CATs, regardless of the substituents on the benzene ring and/or the
alkyl chain length. Cathinone (primary amine) and methcathinone (secondary amine) were
selected as model drugs (Figure 1a), and the two hydrogen atoms eliminated during
dehydration were identified using their deuterium (D)-substituted compounds to elucidate the
dehydrated ion generation mechanism.
The two main metabolites of CATs with a tertiary amine structure, the reductive
metabolites (1-OH metabolite) and oxidative metabolites (2″-oxo metabolite) (Figure 1b),
generate dehydrated ions in ESI-CID. These metabolites lose the cathinone skeleton by
conversion from a carbonyl group to a hydroxy group and an amine group to an amide group,
which leads to the generation of dehydrated ions [6–10,19,20]. In this study, the two
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metabolites of α-pyrrolidinobutiophenone (α-PBP) selected as a model drug of α-PPs, had
hydrogen and oxygen atoms eliminated during dehydration and these were identified using
1
8
stable isotope (D or oxygen-18 ( O))-labeled metabolites synthesized in our laboratory.
2
Experimental
2
.1 Reagents
Cathinone, methcathinone, dimethylcathinone, α-PBP, 1-OH-α-PBP and 2″-oxo-α-PBP
were synthesized according to the previously-reported methods [5,7]. Six stable isotope
labeled compounds (Figure 2) were also synthesized in our laboratory according to the
1
methods described below. All synthesized standards were confirmed by H nuclear magnetic
resonance (NMR) spectroscopy and liquid chromatography-high-resolution mass
spectrometry. Deuterium oxide (99.9 %, for NMR) and methanol-d
4
(≥99.8 atom % D) were
purchased from Wako Pure Chemical Industries (Osaka, Japan) and Sigma-Aldrich Japan
1
8
(Tokyo, Japan), respectively. 2-Pyrrolidinone-3,3,4,4,5,5-d
6
(99.6 %-d
6
) and water- O
1
8
(
≥98 %- O) were obtained from C/D/N Isotopes (Pointe Claire, QC, Canada) and Taiyo
Nippon Sanso (Tokyo, Japan), respectively. All other chemicals and reagents were of
analytical grade and were obtained from Wako. Distilled water and LC/MS-grade methanol
were used throughout the experiments.
2
.2 Synthesis of stable isotope-labeled compounds
2
.2.1 Synthesis of deuterium labeled compounds by H/D exchange method
The D-labeled cathinone (cathinone-d
2
, Figure 2a), D-labeled methcathinone
(methcathinone-d, Figure 2b) and D-labeled 1-OH-α-PBP (1-OD-α-PBP, Figure 2c) were
simply synthesized using a following H/D exchange method: One milligram of each
compound (cathinone, methcathinone, and 1-OH-α-PBP) was dissolve in one milliliter of
methanol-d
4
–deuterium oxide (1:4) solvent to substitute hydrogen atoms bonded nitrogen
atom and oxygen atom with D atoms.
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2
.2.2 1-(1-oxo-1-phenylbutan-2-yl)pyrrolidin-2-one-3,3,4,4,5,5-d
″-Oxo-α-PBP-d was synthesized according to the methods used in our previous study
7] (Figure 3a): One drop of bromine in dichloromethane was added to a dichloromethane
6
(2″-Oxo-α-PBP-d
6
)
2
6
[
solution of butyrophenone (Wako). This solution was stirred for 5 min for the reaction to
initiate. An equimolar amount of bromine solution was added over a period of a further
1
0 min. The solvent was removed under vacuum to yield 2-bromo-butyrophenone. To a
solution of 2-bromobutyrophenone in tetrahydrofuran (THF; Wako) at 0 °C, a suspension of
-pyrrolidinone-3,3,4,4,5,5-d and sodium hydride (60 %, dispersion in paraffin liquid) in
2
6
THF was added dropwise. The mixture was stirred at room temperature for 48 h. The reaction
mixture was treated with 10 % hydrochloride aqueous solution to make it acidic and was then
washed with diethyl ether. The aqueous layer was then made basic with 10 % sodium
carbonate and extracted with ethyl acetate. The organic extract was washed with brine, dried
over anhydrous sodium sulfate, and evaporated under vacuum. The resultant residue was
subjected to column chromatography using a silica gel column and an ethyl acetate/n-hexane
mixture (1:1, v/v) as an eluent to isolate 2″-oxo-α-PBP-d as pale yellow oil.
6
2
.2.3 1-(1-oxo-1-phenylbutan-2-yl-2-d)pyrrolidin-2-one (2″-Oxo-α-PBP-d)
″-Oxo-α-PBP-d was synthesized by reference to the methods of Harbeson [21] as
follows (Figure 3b): To a solution of butyrophenone in methanol-d , deuterium oxide was
2
4
added slowly under nitrogen gas, followed potassium carbonate. The mixture was stirred at
room temperature for 20 h. The mixture was concentrated in vacuo nearly to dryness and then
partitioned between ethyl acetate and deuterium oxide. The organic layer was washed with
saturated sodium chloride deuterium oxide solution, dried over anhydrous sodium sulfate,
and evaporated under vacuum to obtain a colorless oil of butyrophenone-2,2-d
One drop of bromine in dichloromethane was added to a dichloromethane solution of
butyrophenone-2,2-d . This solution was stirred for 5 min for the reaction to initiate. An
2
.
2
equimolar amount of bromine solution was added over a period of a further 10 min. The
solvent was removed under vacuum to yield 2-bromobutyrophenone-2-d.
To a solution of 2-bromobutyrophenone-2-d in THF at 0 °C, a suspension of
2
-pyrrolidinone and sodium hydride (60 %, dispersion in paraffin liquid) in THF was added
dropwise. The mixture was stirred at room temperature for 48 h. The reaction mixture was
treated with 10 % hydrochloride aqueous solution to make it acidic and was then washed with
diethyl ether. The aqueous layer was then made basic with 10 % sodium carbonate and
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extracted with ethyl acetate. The organic extract was washed with brine, dried over anhydrous
sodium sulfate, and evaporated under vacuum. The resultant residue was subjected to column
chromatography using a silica gel column and an ethyl acetate/n-hexane mixture (1:1, v/v) as
an eluent to isolate 2″-oxo-α-PBP-d as pale yellow oil.
1
8
18
2
.2.4 1-(1-(oxo- O)-1-phenylbutan-2-yl)pyrrolidin-2-one (2″-Oxo-α-PBP- O)
1
8
2
″-Oxo-α-PBP- O was synthesized by modifying the methods of Attygalle et al. [22] as
1
8
follows (Figure 3c): To a solution of 2″-oxo-α-PBP in THF, water- O and concentrated
hydrosulfate were added, and the mixture was stirred at 50 °C for 12 h. The mixture was then
made basic with potassium carbonate and extracted with ethyl acetate. The organic extract
was washed with brine, dried over anhydrous sodium sulfate, and evaporated under vacuum
1
8
to obtain a pale yellow oil of 2″-oxo-α-PBP- O.
2
.3 Sample preparation
The stock standard solutions (1 mg/mL) of CATs and metabolites, and their stable isotope
labeled compounds were stored in methanol or methanol-d
4
at −20 °C until analysis, and were
(4:1) solution to
diluted with water-methanol (4:1) or deuterium oxide-methanol-d
4
appropriate concentrations as needed for analysis with the mass spectrometer. The D-labeled
compounds synthesized using an H/D exchange method were invariably stored in deuterium
oxide and methanol-d4, and were diluted with them as needed.
2
.4 Instruments
Analysis was performed on an LTQ XL linear ion-trap mass spectrometer (Thermo Fisher
Scientific, Waltham, MA, USA) equipped with an ESI interface. The single scan (MS) and
2
3
product ion scan (MS and MS ) analysis was conducted in the positive mode, and the
protonated and deuterated molecule of each compound were selected as precursor ions. MS3
spectra of 2″-oxo-α-PBP and 2″-oxo-α-PBP-d
6
were obtained by selecting their dehydrated
+
+
ions ([M+H−H
2
O] ; [M−17] ) as precursor ions. Other conditions were as follows: sheath gas,
nitrogen at flow rate of 35 arbitrary unit; source voltage, 4.0 kV; ion transfer capillary
temperature, 270 °C; capillary voltage, 31 V; tube lens voltage, 100 V; isolation width,
1
.0 Da; damping gas, helium; activation Q, 0.25; activation time 30 ms; and normalized
collision energy, 35 %; wideband activation, disabled; infusion rate, 10 μL/min.
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3
Results and Discussion
3
.1 Generation of dehydrated ions from CATs with primary or secondary amines
In an ion trap instrument, such as the one used in this study, CID is accomplished by
exciting the precursor ions at their resonant frequency. As the product ions have different
mass-to-charge-ratio (m/z) from the precursor ion, they are not in resonance with the
excitation frequency, and are not subjected to further ion fragmentation (single-step
fragmentation). Therefore, the fragment pattern (product ion mass spectrum) obtained is
hardly influenced by collision energy in CID. Under such analytical condition, we examined
+
the generation mechanism of the dehydrated product ion ([M+H−H
2
O] ) from the protonated
+
CATs ([M+H] ). As shown in Figures 4a–4c, cathinone (primary amine) and methcathinone
(secondary amine) both show dehydrated ions (m/z 132 and 146, respectively) as base peaks.
As expected, the tertiary amine, dimethylcathinone, did not produce a dehydrated ion as a
product ion. Considering that only primary and secondary amine-containing structures
produced dehydrated ions, it was predicted, that one of two hydrogen atoms eliminated
during dehydration is bonded to nitrogen (tertiary amines have no hydrogen bonded to
nitrogen). Therefore, the synthesized N-deuterated compounds (Figures 2a and 2b) were
analyzed under the condition of methanol-d
4
-deuterium oxide. As shown in Figures 4d and 4e,
+
each compound produced a product ion (m/z 133 or 146) corresponding to [M+D−D
2
O] as a
+
+
base peak, while ions corresponding to [M+D−HDO] or [M+D−H O] were not detected.
2
These results indicate that the hydrogen atom bonded to nitrogen was eliminated along with
the protons added to the compound during ESI and suggest that CATs with primary and
secondary amines undergo water elimination according to the reaction mechanism shown in
Scheme 1. This finding demonstrates that dehydration does not occur in CATs with a tertiary
amine structure and also shows that the presence or absence of dehydrated ions can be used to
identify the amine structures of CATs.
3
.2 Generation of dehydrated ions from the main metabolites of α-PBP (tertiary amine
structure)
As described above, α-PPs have a tertiary amine structure and do not produce a
dehydrated product ion [6–9,16,17,19,20]. However, dehydrated ions are generated from the
two major metabolites of α-PPs, the 1-OH and 2″-oxo [6–9,19,20]. In this section, the
dehydration mechanism of the metabolites of α-PBP, 1-OH-α-PBP and 2″-oxo-α-PBP (Figure
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1
b), were examined and discussed.
3
.2.1 Dehydration of 1-OH-α-PBP
The product ion spectrum of α-PBP, shown in Figure 5a, did not contain a dehydrated ion
(
m/z 200); however, a dehydrated ion (m/z 202) was observed, as the base peak for
-OH-α-PBP (diastereomers 1 and 2; Figures 5b and 5c). To identify the hydrogen atoms
eliminated during dehydration in 1-OH-α-PBP, 1-OD-α-PBP (Figure 2c) was analyzed with a
1
methanol-d
4
-deuterium oxide solvent. In each case of diastereomers, the product ion
+
corresponding to [M+D−D
2
O] (m/z 202) was observed as a base peak (Figures 5d and 5e),
+
+
while ions corresponding to [M+D−HDO] and [M+D−H O] were not detected. These
2
results indicate that upon dehydration of 1-OH-α-PBPs, the hydrogen atom on the hydroxy
group was eliminated with the proton added during ESI. This suggests that water molecules
were eliminated according to the reaction mechanism shown in Scheme 2. Therefore, the
dehydration reaction of 1-OH metabolites of α-PPs proceeds without hydrogen atoms on the
amine group, as the carbonyl group in the cathinone skeleton is reduced to a hydroxy group.
Similarly, in dimethylcathinone with a tertiary amine structure, the reductive metabolite
(1-OH metabolite, Figure 1b), methylephedrine (antitussive drug), generated a dehydrated ion
+
(
[M+H−H
2
O] ) in ESI-CID by the same dehydration mechanism (results not shown).
When comparing diastereomers 1 and 2, the dehydrated ion of diastereomer 1 (m/z 202)
was observed with a strong intensity on the single scan mass spectrum (Figure 5b MS), which
suggests that dehydration proceeds more readily in diastereomer 1 compared to diastereomer
2
. Our previous study [7] showed that the diastereomer 1 and 2 of 1-OH-α-PBP are syn-form
and anti-form isomers, respectively. The hydroxy group of diastereomer 1 (syn form) is
therefore located in the three-dimensional position closer to the proton added to the amine
group during ESI, compared to that of diastereomer 2 (anti form). The location of the
hydroxyl group could contribute to more rapid dehydration of diastereomer 1.
3
.2.2 Dehydration of 2″-oxo-α-PBP
″-Oxo-α-PBP is an oxidative metabolite of α-PBP, and the oxidation of the 2″ position of
2
pyrrolidine ring changes the structure from an amine to an amide (Figure 1b). In the product
ion spectrum, the dehydration ion (m/z 214) was observed at low intensity (Figure 6a). In
order to identify the oxygen and hydrogen atoms eliminated during dehydration, three
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stable-isotope compounds (Figures 2d–2f) were synthesized and analyzed.
1
8
First, an O-labeled compound (Figure 2f) was analyzed to identify the oxygen atom
eliminated during dehydration. As shown in Figure 6b, the dehydrated ion corresponding to
M+H−H 18
O] was detected at m/z 214. This indicated that the oxygen atom in the alkyl
+
[
2
carbonyl group was eliminated during dehydration, while the pyrrolidone ring was not
involved in the reaction.
The pyrrolidone ring-deuterium labeled compound (Figure 2d) was dissolved in
methanol-water solvent and analyzed to investigate the involvement of the pyrrolidone ring
hydrogen atoms during dehydration. As shown in Figure 6c, the dehydrated ion
+
corresponding to [M+H−H
2
O] was detected at m/z 220, indicating that the hydrogen atoms
on the pyrrolidone ring were not involved in dehydration. In addition, the product ion
spectrum produced from the dehydrated ion (m/z 220) included a pyrrolidone-d -eliminated
ion (m/z 129), suggesting that the pyrrolidone ring was not involved in dehydration (Figure
c MS3).
Additionally, in order to identify which hydrogen atom becomes eliminated, an alkyl
chain-deuterium labeled compound (Figure 2e) was dissolved either in methanol-water
solvent or in methanol- d -deuterium oxide solvent (Figures 6d and 6e). As shown in Figure
d, under methanol-water solvent condition, two dehydrated ions corresponding to
6
6
4
6
+
+
[
M+H−HDO] and [M+H−H
indicating that the hydrogen atom at position 2 of the alkyl chain was partially involved in the
dehydration process. On the other hand, under methanol-d -deuterium oxide solvent
2
O] were detected at m/z 214 and m/z 215, respectively,
4
+
condition, dehydrated ion corresponding to [M+D−HDO] was detected at m/z 215 (Figure
e). The fact that a hydrogen atom was pulled out of a deuterated molecule revealed that a
6
hydrogen atom other than a) the hydrogen atom at position 2 of the alkyl chain and b) the
proton added during ESI was eliminated during the dehydration reaction. Furthermore, to
determine whether the proton added during ESI was involved in the dehydration process,
2
″-oxo-α-PBP dissolved in methanol-d
4
-deuterium oxide solvent was analyzed. The
+
+
dehydrated ions corresponding to [M+H−HDO] (m/z 214) and [M+H−H
2
O] (m/z 215) were
detected as product ions, as shown in Figure 6f, and it indicates that the proton added during
ESI is also partially eliminated during dehydration. Ratios of ion intensity of m/z 214 to that
of m/z 215 shown in Figures 6d and 6f additionally suggested that the proton added during
ESI is likely to be more involved in the dehydration reaction than the hydrogen atom at
position 2 of the alkyl chain. Besides the dehydrated ions, product ions corresponding to
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+
[
M+D−C
4
H NO] , loss of a pyrrolidine ring, were abundantly detected at m/z 149 and
7
m/z 148, shown in Figures 6e and 6f, respectively. It is worth noting that these ions still
contain deuterium atoms after losing the pyrrolidone ring from the deuterated molecule; this
suggests that the deuterium ion added during ESI is exchanged from its initial position (most
likely at the nitrogen atom), presumably moving to the cathinone backbone.
In summary, results from this study demonstrated that three or more hydrogen atoms (the
hydrogen atom at position 2 of the alkyl chain, the proton added during ESI, and others) were
eliminated with an oxygen atom in the alkyl carbonyl group during dehydration, while likely
involving the rearrangement reaction of hydrogen atoms.
4
Conclusion
In this study, the generation mechanism of dehydrated ions for CATs during ESI-CID was
investigated using synthesized stable-isotope labeled compounds. For CATs that have a
primary or secondary amine structure, the hydrogen atom bonded to the nitrogen of the amine
was found to be eliminated with the proton added during ESI and the carbonyl oxygen atom,
which demonstrates that dehydration does not occur in CATs with a tertiary amine structure.
This result provides evidence that dehydrated ions can be used to distinguish amine structures
of CATs. However, the two major tertiary amine metabolites of CATs (1-OH and 2″-oxo)
produce dehydrated ions as product ions. The dehydration of the 1-OH metabolite occurs by a
simple mechanism involving the elimination of a hydroxyl group, while that of the 2″-oxo
metabolite involves hydrogen atoms in three or more locations via multiple mechanisms.
Further research on the intricate dehydration mechanism for 2″-oxo metabolites is in progress.
The findings from this study will assist in the elucidation of reaction mechanisms in ESI-CID
and provide significant contributions to the structural identification of abused drugs,
including CATs.
Acknowledgments
We wish to thank Dr. Hisao Nakata, Professor Emeritus, Aichi Kyoiku University for
advice on fragmentation mechanism.
This work supported by JSPS KAKENHI Grant Number 17H00551 and 20H00966.
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(
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Figure 1 Chemical structures of (a) cathinone (primary amine) and methcathinone (secondary
amine), and (b) dimethylcathinone (tertiary amine), α-PBP (tertiary amine) and their
metabolites.
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1
8
Figure 2 Stable isotope (D or O)-labeled compounds synthesized in this study.
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Figure 3 Synthetic pathways of stable isotope labeled compounds. (a) 2″-Oxo-α-PBP-d , (b)
6
1
8
2
″-Oxo-α-PBP-d, (c) 2″-Oxo-α-PBP- O.
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2
Figure 4 ESI-MS and ESI-CID-MS spectra of (a) cathinone in water-methanol (4:1), (b)
methcathinone in water-methanol (4:1), (c) dimethylcathinone in water-methanol (4:1), (d)
cathinone-d
2
in deuterium oxide-methanol-d
4
(4:1), and (e) methcathinone-d in deuterium
oxide-methanol-d
4
(4:1). Each protonated or deuterated molecule was selected as a precursor
ion.
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2
Figure 5 ESI-MS and ESI-CID-MS spectra of (a) α-PBP in water-methanol (4:1), (b)
1
-OH-α-PBP-diastereomer 1 in water-methanol (4:1), (c) 1-OH-α-PBP-diastereomer 2 in
water-methanol (4:1), (d) 1-OD-α-PBP-diastereomer 1 in deuterium oxide-methanol-d
and (e) 1-OD-α-PBP-diastereomer 2 in deuterium oxide-methanol-d (4:1). Each protonated
or deuterated molecule was selected as a precursor ion.
4
(4:1),
4
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2
3
Figure 6 ESI-MS, ESI-CID-MS and -MS spectra of (a) 2″-oxo-α-PBP in water-methanol
1
8
(
4:1), (b) 2″-oxo-α-PBP- O in water-methanol (4:1), (c) 2″-oxo-α-PBP-d
6
in water-methanol
(4:1), (d) 2″-oxo-α-PBP-d in water-methanol (4:1), (e) 2″-oxo-α-PBP-d in deuterium
oxide-methanol-d
4
(4:1), and (f) 2″-oxo-α-PBP in deuterium oxide-methanol-d (4:1). Each
4
2
protonated molecule was selected as a precursor ion for MS , and the dehydrated ions [M+H–
+
3
H O] were selected as precursor ions for MS .
2
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Scheme 1 Proposed mechanism for generation of the dehydrated ion during ESI-CID for
CATs with primary and secondary amines.
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Scheme 2 Proposed mechanism for generation of the dehydrated ion during ESI-CID for
1
-OH-α-PBP (1-OH-α-PPs).
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