Structural determination of the novel fragmentation routes of zwitteronic morphine opiate...

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2007; 21: 1062–1074

) DOI: 10.1002/rcm.2935
Published online in Wiley InterScience (www.interscience.wiley.com
Structural determination of the novel fragmentation

routes of zwitteronic morphine opiate antagonists

naloxonazine and naloxone hydrochlorides using

electrospray ionization tandem mass spectrometry

Nicolas Joly1, Celine Vaillant2, Alejandro M. Cohen3, Patrick Martin1, Mokhtar El Essassi4,

Mohamed Massoui4 and Joseph Banoub2,3*1Laboratoire de Physico-Chimie des Interfaces et Applications FRE CNRS 2485, Federation Chevreul FR CNRS 2638, Site de Bethune,

IUT de Bethune BP819, 62408 Bethune, France2Department of Chemistry, Memorial University of Newfoundland, St. John’s, Newfoundland, A1B 3V6, Canada3Fisheries and Oceans Canada, Science Branch, Special Projects, P.O. Box 5667, St John’s, Newfoundland, A1C 5X1, Canada4Pole de Competence Pharmacochimie, Laboratoire de Chimie Organique Heterocyclique, Universite Mohammed V-Agdal,

Rabat, Morocco

Received 1 December 2006; Revised 15 January 2007; Accepted 16 January 2007

*Correspoorial UniA1B 3V6,E-mail: bContract/Research

Electrospray ionization quadrupole time-of-flight (ESI-QqToF) mass spectra of the zwitteronic salts

naloxonazine dihydrochloride 1 and naloxone hydrochloride 2, a common series of morphine opiate

receptor antagonists, were recorded using different declustering potentials. The singly charged ion

[MRH–2HCl]R at m/z 651.3170 and the doubly charged ion [MR2H–2HCl]2R at m/z 326.1700 were

noted for naloxonazine dihydrochloride 1; and the singly charged ion [MRH–HCl]R at m/z 328.1541

was observed for naloxone hydrochloride 2. Low-energy collision-induced dissociation tandem

mass spectrometry (CID-MS/MS) experiments established the fragmentation routes of these com-

pounds. In addition to the characteristic diagnostic product ions obtained, we noticed the formation

of a series of radical product ions for the zwitteronic compounds 1 and 2, and also the formation of a

distonic ion product formed from the singly charged ion [MRH–HCl]R of naloxone hydrochloride 2.

Confirmation of the various established fragmentation routes was effected by conducting a series of

ESI-CID-QqTof-MS/MS product ion scans, whichwere initiated by CID in the atmospheric pressure/

vacuum interface using a higher declustering potential. Deuterium labeling was also performed on

the zwitteronic salts 1 and 2, in which the hydrogen atoms of the OH andNH groups were exchanged

with deuterium atoms. Low-energy CID-QqTof-MS/MS product ion scans of the singly charged and

doubly charged deuteriated molecules confirmed the initial fragmentation patterns proposed for the

protonated molecules. Precursor ion scan analyses were also performed with a conventional quad-

rupole-hexapole-quadrupole tandemmass spectrometer and allowed the confirmation of the genesis

of some diagnostic ions. Copyright # 2007 John Wiley & Sons, Ltd.

The opiates are a naturally occurring basic alkaloid series of

compounds, such as morphine, codeine and heroin, which

have a high pharmacological activity, that helps to moderate

pain in several terminal diseases.1 The therapeutically useful

effects and adverse side issues associated with opiate use are

primarily due to interaction with m receptors. Opiates induce

sleep, relieve pain, and cause sedation and pleasure by acting

on the brain’s m, d and k peptide neurotransmitter receptors,

releasing endorphins and encephalins. The excess use of

opiates releases an excess of dopamine in the brain, resulting

in a constant need for the drug to block the activated opiate

receptor site, a phenomenon which causes drug addiction.2

ndence to: J. Banoub, Department of Chemistry, Mem-versity of Newfoundland, St John’s, Newfoundland,Canada.

[email protected] sponsor: Natural Sciences and EngineeringCouncil of Canada.

Eight million people worldwide are addicted to opiate use,

the ‘drug addiction’ syndrome.3 The pharmacotherapy of

opiate addiction is used to target opiate withdrawal

symptoms, to facilitate the initiation of abstinence and/or

to reduce relapse to opiate use, by maintenance on agonist

or antagonist agents. The two major goals of pharmacotherapy

are the relief of opiate withdrawal symptoms, and relapse

prevention, either after abstinence initiation or after being

stabilized on a long-acting opiate agonist such as methadone,

or an antagonist such as naloxone or naloxonazine.4,5 Opioids

can cause respiratory depression at excessive doses or if

administered in the absence of pain, so it is essential that these

agents are titrated against the level of pain experienced.6

Copyright # 2007 John Wiley & Sons, Ltd.

N

OO

N

OHOH

OHO NH N O

N

OH

O OH

+2H++H+

.Cl-.2Cl-

Naloxone hydrochloride 2C19H21NO4.HCl

Mr 363.835

Naloxonazine dihydrochloride 1C38H42N4O6.2HCl

Mr 723.685

Scheme 1. The molecular structures of naloxonazine dihy-

drochloride 1 and naloxone hydrochloride 2.

Fragmentations of zwitteronic morphine opiate antagonists 1063

Various analytical assays have been developed for

pharmacokinetic studies and forensic analysis of different

opiate analgesics. Included in these are immunoassay tests

which are sensitive but lack the specificity to distinguish the

opiates from their corresponding glucuronide metabolites,

which may cross-react with the antisera.7,8

Analyses of opiates by gas chromatography/mass spec-

trometry (GC/MS) and liquid chromatography/mass

spectrometry (LC/MS) methods have been reviewed

recently, but none of the reviews have addressed the analysis

of the antagonists, especially in the presence of the opiates

and their respective metabolites.9,10 Identification of mo-

rphine, codeine and their metabolites was also achieved by

capillary electrophoresis combined with electrospray ioniz-

ation using a quadrupole ion trap (ESI-QIT-MSn).11

We have recently reported the structural determination of

the novel fragmentation routes of morphine opiate receptor

antagonists using electrospray ionization quadrupole time-

of-flight tandem mass spectrometry (MS/MS). Low-energy

collision-induced dissociation (CID)-MS/MS experiments

established the fragmentation routes of these compounds.12

This study was used by our coworkers in the clinical

identification of the metabolites in plasma from subjects

treated with this series of antagonists.

In a continuation of our previous work on these

antagonists, we now report on the structural characterization

of the zwitteronic salts of the naloxonazine dihydrochloride 1

and the naloxone hydrochloride 2 (see Scheme 1), two

important pharmacological antagonists of the m1 and m2

receptors. Preclinical and clinical studies have shown that

co-treatment of morphine with extremely low doses of

opioid antagonists (such as naloxone) attenuates opioid

tolerance and dependence.13 It was also found that the

endogenous opioid system was implicated in excessive

ethanol-drinking behavior. The selective m1-opioid antagon-

ist, naloxonazine, was shown to modulate alcohol-drinking

behavior.14 To our knowledge there have been no previous

studies on the mass spectrometric characterization and

the fragmentation routes of these types of morphine opiate

receptor antagonists using ESI.

EXPERIMENTAL

Sample preparationThe derivatives, naloxonazine dihydrochloride 1 and

naloxone hydrochloride 2, were purchased from Tocris

Cookson (St. Louis, MO, USA). The molecular structures of

these derivatives are shown in Scheme 1.


ESI-MSMass spectrometry was performed using an Applied

Biosystems API QSTAR XL (Foster City, CA, USA) quadru-

pole orthogonal time of-flight (QqToF)-MS/MS hybrid

instrument capable of analyzing a mass range of m/z

5–40 000, with a resolution of 10 000 in the positive ion

mode. ESI was performed with a Turbo Ionspray source

operated at 5.5 kV at a temperature of 808C. Each morphine

antagonist (0.1 mg) was dissolved in methanol/water (10:1)

to achieve a concentration of 0.1 mmol/mL. Aliquots (3 mL)

were infused into the mass spectrometer with an integrated

Harvard syringe pump at a rate of 0.1 mL/min. Calibration

of the ToF analyzer was performed using a standard of

porcine renin substrate tetradecapeptide (Mass Spectrometer

Standards Kit, Applied Biosystems). The monoisotopic peaks

of the [MþH]þ and [Mþ2H]2þ ions at m/z 1758.9326 and

879.9699, respectively, were selected for exact mass cali-

bration of the ToF analyzer.

Low-energy CID-MS/MSProduct ion spectra were obtained arising from fragmenta-

tion in the radio-frequency (RF)-only, LINAC (linear

acceleration pulsar high pressure) equipped, quadrupole

collision cell of the QqToF-MS/MS hybrid instrument.

Nitrogen was used as the collision gas for MS/MS analyses

with collision energies varying between 10 and 45 eV.

Collision energy (CE) and CID gas conditions were adjusted

such that the precursor ion remained abundant.

In addition, re-confirmation of the various established

fragmentation routes was effected by conducting a series of

third-generation ESI-CID-QqTof-MS/MS experiments on the

diagnostic product ions, which were initiated by CID in

the atmospheric pressure/vacuum interface using a higher

declustering potential.

Precursor ion scanning experimentsPrecursor ion scanning experiments were recorded with a

Micromass Quattro quadrupole-hexapole-quadrupole mass

spectrometer, equipped with an ESI source and capable of

analyzing ions up to m/z 4000. In the precursor ion mode, the

second mass resolving quadrupole (Q2) was held at selected

m/z values to measure the occurrence of particular product

ions produced by collision with argon in the (RF-only)

hexapole, while the first quadrupole (Q1) scanned for

the corresponding precursors. A personal computer

(Compaq PIII 500MHz processor, running Windows NT 4,

service pack 3) equipped with Masslynx 3.3 Mass Spec-

trometry Data System software was used for data acquisition

and processing. The temperature of the ESI source was 708C.

RESULTS AND DISCUSSION

ESI-QqToF-MS analyses of naloxonazinedihydrochloride 1 and naloxonehydrochloride 2The positive ion ESI mass spectrum of naloxonazine

dihydrochloride 1 was recorded with a declustering

potential (DP) of 30 V and gave the singly charged

ion [MþH–2HCl]þ at m/z 651.3170 and the doubly charged

ion [Mþ2H–2HCl]2þ at m/z 326.1700 (Fig. 1(A)). Both


DOI: 10.1002/rcm

Figure 1. ESI mass spectra of naloxonazine dihydrochloride 1 recorded with (A) DP¼ 30V and (B) DP¼ 100V.

(C) ESI mass spectrum of the deuteriated naloxonazine analogues (part of the spectrum is shown).

1064 N. Joly et al.

these diagnostic ions were observed when the dihydro-

chloride 1 was neutralized with a base, followed by

acidification.

The ESI mass spectrum of naloxone hydrochloride 2 was

recorded with a DP of 30 V (Fig. 2(A)). In this spectrum we

noticed the formation of the singly charged ion [MþH–HCl]þ

at m/z 328.1541.


Increasing the DP to 100 V enhanced the ‘in-nozzle’

fragmentation of both compounds resulting in the formation

of a series of diagnostic ions which were also formed, as

expected, in the CID-MS/MS analyses of the respective

[MþH–2HCl]þ ions (Figs. 1(B) and 2(B)). The genesis and

the proposed structure of these ions will be discussed in the

following sections.


DOI: 10.1002/rcm

350340330320310300290280270260250240230220210200190180170160150140130120110m/z

0

10

20

30

40

50

60

70

77 328.1541

329.1516

310.1440

327.1485311.1403

[M+H-HCl]+

A

C

B

350340330320310300290280270260250240230220210200190180170160150140130120110100m/z

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

14001475 212.0656

310.1441

268.1402253.1196 328.1539213.0737185.0589161.0653 226.0962199.0683

153.0754 311.1492269.1247254.1220240.1199128.0709 214.0822186.0753167.0825 329.1576115.0656 282.1530209.0766 343.2222296.1310

336.0335.0334.0333.0332.0331.0330.0329.0328.0327.0326.0325.0324.0323.0m/z

0

100

200

300

400

500

600

700

800

900

1000

1095 328.1541

329.1563

330.1598

331.1652327.1525 327.7217 332.1724328.5233326.1450 329.7270

Figure 2. ESImass spectra of naloxone hydrochloride 2 at (A) DP¼ 30 and (B) DP¼ 100V. (C) ESI mass spectrum of

the deuteriated naloxone analogues (part of the spectrum is seen).


ESI-QqToF-MS/MS analyses of naloxonazinedihydrochloride 1Tandem mass spectrometry analyses (low-energy CID-MS/

MS) were conducted to determine the fragmentation route

pathways leading to the formation of the various product

ions observed in the conventional mass spectra obtained

with different fragmentation voltages. The product ion scan

of the singly charged ion [MþH–2HCl]þ at m/z 651.27 was


recorded with a collision energy (CE) of 45 eV and is shown

in Fig. 3(A). The spectra afforded product ions at m/z 633.25,

615.24, 592.22, 574.22, 532.19, 491.12, 405.18, 325.13, 307.12,

295.11 and 292.11 (Fig. 3(A)).

A tentative breakdown pattern12,15–17 of some of this major

series of diagnostic product ions is presented in Scheme 2. In

this validation, the product ion scan of the singly charged ion

[MþH–2HCl]þ1 afforded the ions 1a at m/z 633.25 and 1b at


DOI: 10.1002/rcm

A

C

B

70065060055050045040035030025020015010050m/z

0

20

40

60

80

100

120

140

160

180

200

218 633.25

615.24307.13

325.13292.12 592.22 651.27

254.11 266.09 359.01 405.19242.11 574.22532.19327.15167.0573.05 175.07 491.13 617.2384.08 417.18 635.24

70065060055050045040035030025020015010050m/z

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

26002742 636.23

656.27

618.22

308.12327.13

595.21293.11 638.24255.10 307.11226.08204.09 407.1984.08 363.16 534.18176.07 576.20269.10 418.18 493.17

70065060055050045040035030025020015010050m/z

0

100

200

300

400

500

600

700

800

900

1000

1100

1200308.14

295.13

321.14

317.14

254.11326.14293.12

267.11240.09 277.12222.1270.07 84.08 310.15 339.15187.04

Figure 3. (A) ESI-CID-MS/MS of selected ion [MþH–2HCl]þ atm/z 651.31 isolated from naloxonazine hydrochloride

using CE¼ 45 eV, (B) ESI-CID-MS/MS of the singly charged precursor monomer pentadeuteriated product ion atm/z

656.27 (naloxonazine hydrochlorideþCH3OD). (C) Product ion scan of the [Mþ2H–2HCl]2þ ion at m/z 326.14.

1066 N. Joly et al.

615.24, by the respective elimination of either one or two

molecules of water. Fission of the N–N single bond afforded

the charged monomer 1h at m/z 325.13. This latter ion

eliminated a molecule of water to produce 1i at m/z 307.12.

An extremely interesting and surprising finding was the

formation of the radical ions 1c, 1d, 1f and 1g, at m/z 592.22,

574.22, 532.19 and 405.18, respectively. This series of product

radical ions was tentatively assigned as follows: 1c at m/z

592.22 was formed by either a consecutive or a concerted

elimination of a molecule of water, and a 1,2-propylene


radical ion, or vice versa. The ion 1d, at m/z 574.2,2 was

obtained, in a similar manner to the former ion, by

elimination of two molecules of water and a 1,2-propylene

radical ion. Similarly the ion 1f at m/z 532.19 was obtained

by either the consecutive or stepwise elimination of two

molecules of water, a molecule of 1,2-propylene and a

propylene radical ion, not necessarily in that order. Finally

the ion 1g at m/z 405.18 was obtained by a series of either

consecutive or alternate eliminations of two molecules

of water and a 1,2-propylene radical, followed by two


DOI: 10.1002/rcm

- H2O- 2 H2 H-O 2O

- H2C=CH-CH2.

- 2 H2O- H2C=CH-CH2

.- 2 H2O

- H2C=CH-CH2.

N-

- 2 H2O- H2C=CH-CH2

.

- H2C=CH-CH3 O

N

HO

HN OH

-- 2 H2O

- H2C=CH-CH2.

- 2 HC≡C-OH

N-

- H2O

O

N

NHO O

N

HO

N OH

C38H41N4O5m/z 633.25

+ H

+

Deuterated molecules634.22; 635.22; 636.23

O

N

NHO O

N

N OH

C38H39N4O4m/z 615.24

+ H

+



O

N

NHO O

N

N OH

C32H28N4O4m/z 532.19

++H

O

N

NHHO

C19H19N2O2m/z 307.12

+

Deuterated molecules308.11; 309.11


O

N

NHO O

N

HO

N OH

+

C35H36N4O5m/z 592.22

+H

1

1a1b 1c

1d 1e

1f

1g

1h1i

[C17H15N2O3]+

m/z 295.11

[C19H18NO2]+

m/z 292.11

O

N

OH

NHO O

N

HO

N OH

C38H43N4O6m/z 651.27

+ H

+

Deuterated molecules652.26; 653.26; 654.26;

655.26; 656.26

O

N

OH

NHO

C19H21N2O3m/z 325.13


O

N

NHO O

N

N OH

C35H34N4O4m/z 574.22

++H


O N O

N

N

C26H19N3O2m/z 405.18

+H +


O NHO O

N

N OH

C30H25N3O4m/z 491.12

+H

+


- H2O- 2 H2 H-O 2O

- H2C=CH-CH2.

- 2 H2O- H2C=CH-CH2

.- 2 H2O

- H2C=CH-CH2.

N-

- 2 H2O- H2C=CH-CH2

.

- H2C=CH-CH3 O

N

HO

HN OH

-- 2 H2O

- H2C=CH-CH2.

- 2 HC≡C-OH

N-

- H2O

O

N

NHO O

N

HO

N OH

C38H41N4O5m/z 633.25

+ H

+


O

N

NHO O

N

N OH

C38H39N4O4m/z 615.24

+ H

+



O

N

NHO O

N

N OH

C32H28N4O4m/z 532.19

++H

O

N

NHHO

C19H19N2O2m/z 307.12

+


593.19; 594.20; 595.20

O

N

NHO O

N

HO

N OH

+

C35H36N4O5m/z 592.22

+H

1

1a1b 1c

1d 1e

1f

1g

1h1i

[C17H15N2O3]+

m/z 295.11

[C19H18NO2]+

m/z 292.11

O

N

OH

NHO O

N

HO

N OH

C38H43N4O6m/z 651.27

+ H

+

Deuterated molecules652.26; 653.26; 654.26;

655.26; 656.26

O

N

OH

NHO

C19H21N2O3m/z 325.13


O

N

NHO O

N

N OH

C35H34N4O4m/z 574.22

++H


O N O

N

N

C26H19N3O2m/z 405.18

+H +


O NHO O

N

N OH

C30H25N3O4m/z 491.12

+H

+


Scheme 2. Proposed overall fragmentation pathways obtained from the [MþH–2HCl]þ ion atm/z 651.31 and the product

ion scans of various selected intermediate ions isolated from naloxonazine dihydrochloride 1.


retro-Diels-Alder (RDA) reactions which eliminate mol-

ecules of ethynol (CHCOH, 42 Da) and, finally, the loss of a

molecule of N-propylene aziridine (83 Da). The product

radical ion 1e at m/z 491.12 was assigned as having been

formed from the precursor ion 1 by the consecutive losses of

two molecules of water; a propylene radical and a molecule

of N-propylene aziridine (see Scheme 2). Note that the

formation of radical ions is not new. In 1994, to our

knowledge, we were the first authors to report the formation

of a radical ion from a positively charged even-electron

product ion, in the ESI-CID-MS/MS study of a series of

synthetic difuranic diamine dihydrochlorides containing the

bis(5-aminomethyl- 2-furyl) unit.18 Other authors have

reported similar free radical formation in the ESI process.19,20

To verify the accuracy of the fragmentation routes

obtained during the CID analysis, we exchanged the labile

hydrogen atoms from the corresponding four hydroxyl

groups, with the deuterium atom of CH3OD, prior to

injection into the ESI source. We expected to obtain at least

five different deuteriated molecules which had incorporated

one to four atoms of deuterium together with one deuterium

atom which should have replaced the hydrogen atom of the

positively charged nitrogen groups. It is evident that, since

there are four nitrogen atoms present, we do not know which

one is the one that is charged. As expected, in the ESI-MS

analysis of the deuteriated naloxonazine dihydrochloride 1

we noticed the incorporation of five deuterium atoms,

accounting for the main ions at m/z 652.3207, 653.3248,

654.3290, 655.3336 and 656.3386, in addition to a minor ion at


m/z 657.3397, which may arise from the C-13 isotopic

distributions (see Fig. 1(C)). Second-generation product ion

scans of the selected singly charged precursor monomer

deuteriated ions, as [C38H41N4O6DþH]þ, [C38H40N4O6D2

þH]þ, [C38H39N4O6D3þH]þ, [C38H38N4O6D4þH]þ and [C38

H37N4O6D5þH]þ, at m/z 652.26, 653.26, 654.27, 655.27 and

656.27, respectively, were recorded. For the sake of brevity,

only the CID-MS/MS spectrum of the pentadeuteriated ion

at m/z 656.27 is shown in Fig. 3(B). Careful study of these

CID-MS/MS spectra confirmed the majority of the proposed

structures of the product ions described in Scheme 2, in

which the masses of the various deuteriated product ions are

shown beneath the non-labeled product ions. It is evident

that the masses of the diagnostic product ions have increased

with the expected number of deuterium atoms, thus

confirming the genesis of formation of all the product ions.

It is also apparent that these spectra also contained isotopic

abundances of C-13 along with the addition caused by the

deuterium traveling process, a factor which sometimes

complicates these assignments.

A third-generation product ion scan of the selected

‘intermediate’ singly charged ion 1h at m/z 325.13, obtained

from the product ion mass spectrum of the [MþH–2HCl]þ

ion, afforded a series of product ions assigned as 1i, 1j, 1k, 1l,

1m, 1n, 1o, 1p, 1q, 1r and 1s, at m/z 307.11, 296.11, 283.08,

268.09, 256.08, 242.08, 213.08, 202.07, 187.06, 175.06 and

161.05, respectively (Fig. 4(A) and Scheme 3). The ion 1i at

m/z 307.11 resulted from simple elimination of a water

molecule. The ion 1j at m/z 296.11 was produced by either the


DOI: 10.1002/rcm

A

B3403203002802602402202001801601401201008060

m/z

0

20

40

60

80

100

120

140

160

180

200

220325.13

175.07

202.08

307.13283.09161.05

173.0570.06 242.10151.08 268.09187.07 213.08226.08157.06129.07 296.10256.08 280.11125.0795.0583.06 199.06171.0768.05 342.97

3403203002802602402202001801601401201008060m/z

0

5

10

15

20

25

30

35

40

42 328.12

204.07177.07 309.11

203.07

300.11 310.11286.0984.08 163.06 227.0670.06 268.08189.06 241.08254.08201.06 327.69213.06148.07 186.0696.07

Figure 4. Third-generation product ion scans of (A) the singly charged 1h intermediate ion atm/z 325.13 and (B) the

deuteriated precursor ion 1h at m/z 328.12.

1068 N. Joly et al.

concerted or consecutive losses of a molecule of hydrogen

cyanide and a molecule of hydrogen. The ion 1k at m/z 283.08

was produced by a McLafferty rearrangement involving the

loss of a molecule of propylene. The ion 1l at m/z 268.09 was

formed by the concerted losses of a molecule of hydrogen, a

molecule of hydrogen cyanide and a molecule of ethylene,

not necessarily in that order. The ion 1m at m/z 256.08 was

produced by consecutive elimination of molecules of

hydrogen cyanide and ethylene. The ion 1n at m/z 242.09

originated from the consecutive elimination of molecules of

hydrogen cyanide and propylene. The ion 1o at m/z 213.08

was formed by the complex elimination of a molecule of

ethylene, followed by a McLafferty rearrangement involving

the loss of a molecule of propylene and a RDA rearrange-

ment involving the loss of ethynol. The ion 1p at m/z 202.07

was produced by the consecutive losses of two molecules of

water, two molecules of hydrogen and a molecule of

N-propylene aziridine. The ion 1q at m/z 187.06 was

produced by a series of concerted and/or consecutive

mechanisms, which involved two McLafferty rearrange-

ments involving the losses of a molecule of ethylene and

propylene, followed by two RDA reactions involving the


losses of a molecule of acetylene and a molecule of ethynol.

Finally the ion 1r at m/z 175.06 was formed by the complex

elimination of two molecules of water, two molecules of

hydrogen and a molecule of hydrogen cyanide followed by

the loss of a molecule of N-propylene aziridine.

As before, verification of the fragmentation routes derived

from the CID-MS/MS analysis was conducted using the

deuteriated precursor ion. As expected, we noticed in the

conventional ESI spectra the formation of the corresponding

deuteriated [C19H20NO4DþH]þ, [C19H19NO4D2þH]þ and

[C19H18NO4D3þH]þmolecules at m/z 329,1563, 330,1598 and

331,1652, which corresponded to the introduction of one to

three deuterium atoms. The product ion spectra of these ions

gave a series of ions, in which the majority of the masses have

increased with the expected incorporation of the suitable

number of deuterium atoms, hence confirming the proposed

data in Scheme 3. The product ion spectrum of the

deuteriated ion at m/z 328.12 is shown in Fig. 4(B). Although

all the masses of the characterized diagnostic product ions

have increased in accordance with the structures described in

Scheme 3, there were some inconsistencies regarding some

ions. It is very significant to mention that the masses of the


DOI: 10.1002/rcm

- H2O

- H2

- HCN- H2C=CH2

- H3C-CH=CH2

- H2

- HCN

- HCN- H3C-CH=CH2

N-

- H2C=CH2

- H3C-CH=CH2

- HC≡C-OH

- H2C=CH2

- H3C-CH=CH2

- 2 H2O- 2 H2

-HC≡C-OH- HC≡CH

N-

- 2 H2O - 2 H2

- HCN

N-

- 2 H2O- HCN

N-

1i

1h

O

N

OH

HO

C18H18NO3m/z 296.11


O

C13H3Om/z 175.06

Deuterated molecules176.05

O

N

OH

NHO

C16H15N2O3m/z 283.08


O

N

OH

HO

C16H14NO3m/z 268.09


O

N

OH

HOC15H14NO3m/z 256.08


O

NH

OH

N

C12H9N2O2m/z 213.08


O NH

C14H4NOm/z 202.07


1j

1k

1l

1m

1n1o

1p

1q

1r

1s

O

N

NHO

C19H19N2O2m/z 307.11


O

OH

NHO

C14H12NO3m/z 242.08


C13H5m/z 161.05

+

O

N

OH

NHO

C19H21N2O3m/z 325.13


O

NH

OH

N

C10H7N2O2m/z 187.06


- H2O

- H2

- HCN- H2C=CH2

- H3C-CH=CH2

- H2

- HCN

- HCN- H3C-CH=CH2

N-

- H2C=CH2

- H3C-CH=CH2

- HC≡C-OH

- H2C=CH2

- H3C-CH=CH2

- 2 H2O- 2 H2

-HC≡C-OH- HC≡CH

N-

- 2 H2O - 2 H2

- HCN

N-

- 2 H2O- HCN

N-

1i

1h

O

N

OH

HO

C18H18NO3m/z 296.11


O

C13H3Om/z 175.06

Deuterated molecules176.05

O

N

OH

NHO

C16H15N2O3m/z 283.08


O

N

OH

HO

C16H14NO3m/z 268.09


O

N

OH

HOC15H14NO3m/z 256.08


O

NH

OH

N

C12H9N2O2m/z 213.08


O NH

C14H4NOm/z 202.07


1j

1k

1l

1m

1n1o

1p

1q

1r

1s

O

N

NHO

C19H19N2O2m/z 307.11


O

OH

NHO

C14H12NO3m/z 242.08


C13H5m/z 161.05

+

O

N

OH

NHO

C19H21N2O3m/z 325.13


O

NH

OH

N

C10H7N2O2m/z 187.06


Scheme 3

Scheme 3. Proposed overall fragmentation pathways obtained from the singly charged intermediate product ion 1h at

m/z 325.13 isolated from naloxonazine dihydrochloride 1.


product ions at m/z 175.06 and 161.05 have increased by one

and/or two deuterium atoms, whereas the masses of these

product ions should have remain unchanged. We can

postulate that the increase of one deuterium atom may

result from the substitution of a labile proton situated in a

vicinal a-position to a positively charged center. However,

the increase of two deuterium atoms can most probably be

attributed to ‘deuterium traveling’.

The low-energy product ion spectrum of the doubly

charged ion 10, [Mþ2H–2HCl]2þ at m/z 326.14, selected from

naloxonazine dihydrochloride 1, afforded a series of product

ions assigned as 1t, 1u, 1v, 1w, 1x, 1y, 1z, 1za and 1zb, at m/z

317.13, 308.13, 295.12, 294.13, 277.12, 267.10, 254.10, 240.09

and 224.09, respectively (Fig. 3(C)). The ion 1t at m/z 317.13

was formed by the loss of a molecule of water, while the ion

1u at m/z 308.13 was formed by the loss of two molecules of

water from the precursor ion. The ion 1v at m/z 295.12 was

formed by the loss of individual molecules of water,

hydrogen and propylene by a McLafferty rearrangement.

The ion 1w at m/z 294.13 was produced by the loss of two

molecules of water and a molecule of ethylene. The ion 1x at

m/z 277.12 was formed from the loss of three molecules of

water, a molecule of hydrogen and a molecule of propylene.

The ion 1y at m/z 267.10 was produced by the loss of two

molecules of water, a molecule of propylene and a molecule

of propyne. The ion 1z at m/z 254.10 was formed by the loss of

two molecules of water, a molecule of ethylene and two

molecules of propyne. The ion 1za at m/z 240.09 was


produced by the loss of two molecules of water, two

molecules of ethylene and two molecules of propyne. Finally,

the ion 1zb at m/z 224.09 was produced from the loss of

two molecules of water and two molecules of N-propylene

aziridine, followed by the loss of a molecule of hydrogen.

The proposed mode of formation of this series of diagnostic

product ions, produced in the CID-MS/MS of the doubly

charged ion [Mþ2H–2HCl]2þ at m/z 326.15, is presented in

Scheme 4.

Note that Schemes 2 and 3 are the summary of the overall

fragmentation patterns deduced for the various MS/MS

experiments, illustrating the various product ion scans of the

[MþH–HCl]2þ and [Mþ2H–2HCl]2þ ions and their derived

intermediate product ions selected for naloxonazine dihy-

drochloride 1. In addition, to avoid confusion, it is crucial to

understand that, throughout this manuscript, and in the

following proposed schemes, the masses of the product ions,

which have been underlined, correspond to the masses of the

selected precursor ions used for the additional CID-MS/MS

analyses. Also, the masses of the resulting product ions

originate from the last recorded product ion scan.

Precursor ion scans of some selected ions ofnaloxonazine dihydrochloride 1The precursor ion scans of the product ions at m/z 633, 615,

325, 307 and 240 were also recorded with a quadrupole-

hexapole-quadrupole MS/MS instrument. The precursor ion

scan of the ion 1a at m/z 632 indicated that it was formed from


DOI: 10.1002/rcm

- H2O- 2 H2O

- H3C-CH=CH2- H2O-H2

- 3 H2O- H2

- H3C-CH=CH2

- 2 H2O- 2 H2C=CH2

- 2 H3C-C≡CH

- 2 H2O- H3C-CH=CH2

- H3C-C≡CH

-2 H2O- H2C=CH2- 2 H3C-C≡CH

- 2 H2O

N- 2

- H2

- 2 H2O- H2C=CH2

O

N

NHO O

N

HO

N OH

C38H42N4O5m/z 317.13

+ 2H2 +

O

N

NHO O

N

N OH

C38H40N4O4m/z 308.13

+ 2H2 +

O

N

N O

N

HO

N

C35H30N4O3m/z 277.12

+ 2H2 +

O

NH

NHO O

N

N OH

C32H30N4O4m/z 267.10

+ 2H2 +

O

N

NHO O

N

N OH

C36H36N4O4m/z 294.13

+ 2H2 +

O

NH

NHO

+ 2H

O

HN

N OH

C28H24N4O4m/z 240.09

O

NH

NHO

2++ 2H

O

HN

N OH

C30H28N4O4m/z 254.10

O NHO ON OH

C28H20N2O4m/z 224.09

+ 2H 2 +

O

N

NHO O

N

HO

N OH

C35H34N4O5m/z 295.12

+ 2H2 +

1’

1t1u

1v

1w 1z

1y1x

1za

1zb

O

N

OH

NHO O

N

HO

N OH

C38H44N4O6m/z 326.14

+ 2H2 +

Deuterated molecules327.13; 328.13; 329.14; 330.14; 331.15; 332.12

- H2O- 2 H2O

- H3C-CH=CH2- H2O-H2

- 3 H2O- H2

- H3C-CH=CH2

- 2 H2O- 2 H2C=CH2

- 2 H3C-C≡CH

- 2 H2O- H3C-CH=CH2

- H3C-C≡CH

-2 H2O- H2C=CH2- 2 H3C-C≡CH

- 2 H2O

N- 2

- H2

- 2 H2O- H2C=CH2

O

N

NHO O

N

HO

N OH

C38H42N4O5m/z 317.13

+ 2H2 +

O

N

NHO O

N

N OH

C38H40N4O4m/z 308.13

+ 2H2 +

O

N

N O

N

HO

N

C35H30N4O3m/z 277.12

+ 2H2 +

O

NH

NHO O

N

N OH

C32H30N4O4m/z 267.10

+ 2H2 +

O

N

NHO O

N

N OH

C36H36N4O4m/z 294.13

+ 2H2 +

O

NH

NHO

+ 2H

O

HN

N OH

C28H24N4O4m/z 240.09

O

NH

NHO

2++ 2H

O

HN

N OH

C30H28N4O4m/z 254.10

O NHO ON OH

C28H20N2O4m/z 224.09

+ 2H 2 +

O

N

NHO O

N

HO

N OH

C35H34N4O5m/z 295.12

+ 2H2 +

1’

1t1u

1v

1w 1z

1y1x

1za

1zb

O

N

OH

NHO O

N

HO

N OH

C38H44N4O6m/z 326.14

+ 2H2 +

Deuterated molecules327.13; 328.13; 329.14; 330.14; 331.15; 332.12

Scheme 4. Proposed overall fragmentation pathways obtained from the [Mþ2H–2HCl]2þ ion at m/z 326.14 isolated

from naloxonazine dihydrochloride 1.

1070 N. Joly et al.

the quasi protonated molecule. The precursor ion scan of the

ion 1b at m/z 615 indicated that it could be formed from either

the quasi diprotonated molecule at m/z 326 or the quasi

protonated molecule at m/z 651. The precursor ion 1h at m/z

325 was formed solely from the quasi protonated molecule at

m/z 651, confirming our initial assignment. The ion 1j at m/z

307 was formed from the ions at m/z 325, 633 and 651. It

is logical, therefore, to deduce that the multiple origins of

this series of product ions, obtained in the various CID-MS/

MS experiments and precursor ion scans, arose by either

consecutive or concerted losses. In this context, please note

that ‘consecutive’ or ‘concerted’ losses of the many product

and precursor ions described above and throughout the

whole manuscript, in the CID-MS/MS experiments, simply

means that they are both lost at the same time and within the

same reaction region inside the collision cell of the QqToF

hybrid tandem mass spectrometer. Therefore, it is practically

impossible to deduce the order of elimination involving

‘consecutive’ or ‘concerted’ losses under these conditions of

MS/MS. For the sake of brevity these precursor scans are not

shown here.

ESI-QqToF-MS/MS analyses of naloxonehydrochloride 2The product ion spectrum of the singly charged ion

[MþH–HCl]þ at m/z 328.13, obtained from the protonated

molecule of the zwitteronic naloxone hydrochloride 2,

afforded a series of product ions at m/z 310.11, 268.11,

253.09, 227.06 and 212.05, assigned as the ions 2a, 2b, 2c, 2d

and 2e, respectively (Fig. 5(A)).


The ion 2a at m/z 310.11 was produced by the loss of a

molecule of water from the precursor ion. The ion 2b at m/z

268.11 was formed from the precursor ion 2a by a McLafferty

rearrangement involving the loss of a molecule of propylene.

The ion 2c at m/z 253.09 was formed from the loss of a

molecule of propylimine (57 Da) from the ion 2a. The ion 2d

at m/z 227.06 was formed, from the ion 2b at m/z 268.11, by the

elimination of a molecule of 1H-azirine (41 Da). The ion 2e at

m/z 212.06 was formed from the ion 2b at m/z 268.11, by two

consecutive reactions: a McLafferty rearrangement involving

the loss of a molecule of ethylene and the loss of a molecule of

carbon monoxide, followed by ring contraction, or vice versa.

The various tentative fragmentation routes of the CID-MS/

MS of the precursor singly charged ion [MþH–HCl]þ ion at

m/z 328.16 are shown in Scheme 5.

Third-generation product ion scans (also called quasi MS3)

of the individually selected singly charged product ions at

m/z 310.11, 268.11, 253.11, 227.07 and 212.07, produced by

increasing the DP values, thereby enhancing the CID nozzle

fragmentation, were recorded and produced a series of

diagnostic product ions which confirmed the initial frag-

mentation routes obtained from the original CID-MS/MS

experiments.

CID-MS/MS of the singly charged ion [MþH–HCl-

H2O]þ2a at m/z 310.11 afforded the product ions at m/z

292.11, 282.12, 268.11, 253.09, 240.11, 227.06, 212.05, 199.06,

181.07 and 173.05, assigned as 2f, 2g, 2b, 2c, 2h, 2d, 2e, 2i, 2j

and 2k, respectively (Fig. 5(B)). The product ions at m/z

268.11, 253.09, 227.06 and 212.05 have already been identified

for the CID-MS/MS of the singly charged ion [MþH–HCl]þ


DOI: 10.1002/rcm

A

B

400380360340320300280260240220200180160140120100m/z

0

50

100

150

200

250

300

350

400

450

500310.11

328.13253.09 268.11

212.05227.06173.05 199.06161.05 251.07185.05 292.11264.08

400380360340320300280260240220200180160140120100m/z

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

749 310.11

268.11

253.09227.06212.05173.05 199.06161.05 251.07187.08 292.11282.12153.06

Figure 5. (A) ESI-CID-MS/MS spectrum of the [MþH–HCl]þ precursor ion at m/z 328.13 isolated from naloxone

hydrochloride 2 and (B) third-generation product ion scans of the singly charged ion at m/z 310.13.


shown in Scheme 5. In this third-generation product ion scan,

the ion 2f at m/z 292.11 was formed by the loss of a molecule

of water from the precursor ion 2a. The ion 2g at m/z 282.12

was formed by a McLafferty rearrangement involving the

loss of a molecule of ethylene from the precursor ion. The ion

2h at m/z 240.11 is formed from the precursor ion by the

consecutive losses of molecules of ethylene and propylene, or

vice versa. This latter ion 2h may lose a molecule of carbon

monoxide followed by ring contraction to afford the ion 2e at

m/z 212.05.

The CID-MS/MS of the singly charged [MþH–2HCl–

H2O–C2H4]þ ion 2g at m/z 282.12 (Fig. 6(A)) afforded the

product ions at m/z 264.09, 254.11, 252.09, 240.08, 238.13,

226.06, 224.08, 212.06 and 198.08, assigned, respectively, as 2l,

2m, 2n, 2h, 2o, 2q, 2p, 2q, 2e and 2r. The ions at m/z 240.08 and

212.06 have already been identified in the CID-MS/MS

spectra of the precursor ions 2 and 2a at m/z 328.16 and 310.11

shown in Scheme 5. The ion 2l at m/z 264.09 was formed by

elimination of a molecule of water from the precursor ion 2g.

The ion 2m at m/z 254.11 originated by the loss of a molecule

of carbon monoxide from the precursor ion. To complicate

matters further we have noticed that the ion 2e0 at m/z 212.05

can also be produced from the ion at m/z 254.11, by a RDA

reaction with the loss of a molecule of ethynol.


The loss of a molecule of hydrogen, from the product ion

2m at m/z 254.11, affords the ion 2n at m/z 252.09. The ion 2n

loses a molecule of ethylene to produce the ion 2q at m/z

224.08. Meanwhile, the ion 2m at m/z 254.11 loses a molecule

of ethylene to afford the ion 2p at m/z 226.06. The ion 2p loses

an imine radical (CH2¼N., 28 Da) to afford the radical ion 2r

at m/z 198.08. The ion 2h at m/z 240.08 was formed from the

precursor ion 2g by a McLafferty rearrangement involving

the loss of a molecule of propylene. The ion 2o at m/z 238.13 is

formed from the ion at m/z 240.08 by the loss of a molecule of

hydrogen occurring by an oxidation process.

Third-generation CID-MS/MS of the singly charged ion

[MþH–HCl–H2O–C3H6]þ2b at m/z 268.11 (Fig. 6(B)) afforded

the product ions at m/z 253.09, 240.10, 227.07, 226.07, 212.05,

211.05, 199.06 and 184.05. The newly formed product ions at

m/z 211.05, 199.07 and 184.0 were assigned as the structures

2s, 2t and 2u, respectively, and are shown in Scheme 5. The

ion 2d at m/z 227.07 eliminates a molecule of carbon

monoxide to afford the ion 2t at m/z 199.07. We have

assigned the radical ion 2s at m/z 211.05 as being formed by

the loss of a hydrogen radical from the ion 2e at m/z 212.05.

This latter ion 2e forms the ion 2u at m/z 184.06 by the loss of

an imine radical (CH2¼N., 28 Da) followed by ring

contraction.


DOI: 10.1002/rcm

OOHO

C14H11O3m/z 227.07

++ H

OO

C12H8O2m/z 184.07

++ H

O

HN

HO

C16H14NO2m/z 252.09

+

+ H

O

N

HO

C14H10NO2m/z 224.08

+

+ H

OHO

C11H9O2m/z 173.04

++ H

O

NH

OHO

C14H8NO3m/z 238.13

+

+ H

O

HN

C14H14NOm/z 212.06

+

+ H

O

N

OH

OHO

[M + H - HCl]+

C19H22NO4

m/z 328.16

+

+ H

OOHO

C14H11O3m/z 227.06

++ H

O

HN

O

C17H14NO2m/z 264.04

+

+ H

2

2a

2b

2e

2d

2t

2f

2g

2h

2l

2m

2n 2o

2p

2q

2e’’

2c

2d

2e’

2k

-H2O

-H2O

-CO

-H2C=CH2

-CO

-CO

-H2O

-HC≡C-OH

-H2C=CH2

-H2C=CH2

-H2C=CH2

- H2C=CH2

- H2C=CHCH3

- H2C=CHCH3

- HN=CHCH2CH3

- H2C=CHCH3

-H2C=CH2

NH-

-H2C=N•

-H2C=N•

-H2

-H•

-HC≡C-OH-HC≡CH

-H•

-HC≡C-OH+H•

-CO

-H2

- HN=CHCH2CH3

2u’

2r

2u2s

N H-

C13H8Om/z 180.08

-H2O

O

HN

OHO

C17H16NO3m/z 282.12

+

+ H

O

N

OHO

C19H20NO3m/z 310.11

+

+ H

O

NH

OHO

C14H10NO3m/z 240.08

+

+ H

O

N

OHO

C16H14NO3m/z 268.11

++ H

OOHO

C16H13O3m/z 253.09

++ H

O

N

O

C19H18NO2m/z 292.11

+

+ H

O

NH

HO

C13H10NO2m/z 212.05

+

+ H

OHO

C13H11O2m/z 199.06

++ H

O

N

HO

C14H12NO2m/z 226.06

++ H

OHO

C13H10O2m/z 198.08

++ H

O

HN

HO

C16H16NO2m/z 254.11

+

+ H

OHO

C12H8O2m/z 184.05

++ H

O

NH

HO

C13H9NO2m/z 211.05

+

+ H

OO

C14H12O2m/z 212.05

++ H

OOHO

C14H11O3m/z 227.07

++ H

OO

C12H8O2m/z 184.07

++ H

O

HN

HO

C16H14NO2m/z 252.09

+

+ H

O

N

HO

C14H10NO2m/z 224.08

+

+ H

OHO

C11H9O2m/z 173.04

++ H

O

NH

OHO

C14H8NO3m/z 238.13

+

+ H

O

HN

C14H14NOm/z 212.06

+

+ H

O

N

OH

OHO

[M + H - HCl]+

C19H22NO4

m/z 328.16

+

+ H

OOHO

C14H11O3m/z 227.06

++ H

O

HN

O

C17H14NO2m/z 264.04

+

+ H

2

2a

2b

2e

2d

2t

2f

2g

2h

2l

2m

2n 2o

2p

2q

2e’’

2c

2d

2e’

2k

-H2O

-H2O

-CO

-H2C=CH2

-CO

-CO

-H2O

-HC≡C-OH

-H2C=CH2

-H2C=CH2

-H2C=CH2

- H2C=CH2

- H2C=CHCH3

- H2C=CHCH3

- HN=CHCH2CH3

- H2C=CHCH3

-H2C=CH2

NH-

-H2C=N•

-H2C=N•

-H2

-H•

-HC≡C-OH-HC≡CH

-H•

-HC≡C-OH+H•

-CO

-H2

- HN=CHCH2CH3

2u’

2r

2u2s

N H-

C13H8Om/z 180.08

-H2O

O

HN

OHO

C17H16NO3m/z 282.12

+

+ H

O

N

OHO

C19H20NO3m/z 310.11

+

+ H

O

NH

OHO

C14H10NO3m/z 240.08

+

+ H

O

N

OHO

C16H14NO3m/z 268.11

++ H

OOHO

C16H13O3m/z 253.09

++ H

O

N

O

C19H18NO2m/z 292.11

+

+ H

O

NH

HO

C13H10NO2m/z 212.05

+

+ H

OHO

C13H11O2m/z 199.06

++ H

O

N

HO

C14H12NO2m/z 226.06

++ H

OHO

C13H10O2m/z 198.08

++ H

O

HN

HO

C16H16NO2m/z 254.11

+

+ H

OHO

C12H8O2m/z 184.05

++ H

O

NH

HO

C13H9NO2m/z 211.05

+

+ H

OO

C14H12O2m/z 212.05

++ H

Scheme 5. Proposed overall fragmentation pathways obtained from the [MþH–HCl]þ ion atm/z 651.31 and the product

ion scans of various selected intermediate ions isolated from naloxone hydrochloride 2.

1072 N. Joly et al.

CID-MS/MS of the singly charged ion [MþH–HCl–

H2O–C3H6]þ2c at m/z 253.09 (Fig. 6(C)) afforded the product

ions at m/z 212.05 (base peak) and 184.07. The ion at m/z

212.05 is formed from the precursor ion 2c at m/z 253.10 by

the loss of 41 Da. We suggest that the formation of the

product radical ion [C14H13O2]þ� at m/z 212.05 occurs by the

neutral loss of an ethynol (CHCOH) molecule, by a RDA

degradation, combined with a hydrogen radical transfer. The

formation of this radical ion, which we assign as a b-distonic

ion, probably arises by a reduction–oxidation mechanism

involving the change of a triple bond into a double bond. This

complex process must occur by an ion–molecule reaction

inside the LINAC collision cell of the QqToF-MS/MS hybrid

instrument. We are aware that this observation does not hold

with the published tenets pertaining to ESI-MS;21 however,

to our knowledge, no one has ever reported such complex

processes inside the collision cell during MS/MS analysis

(Scheme 5).

Holmes originally defined and introduced the term

‘distonic’ ions22 in 1985, followed by Yates, Bouma and

Radom.23 By definition distonic radical ions are a type of ion

in which the positive charge and the radical sites are

separated. Distonic radical ions formally appear from the

ionization of diradicals or zwitteronic molecules (including


ylides).24,25 The product ion 2u0 was formed from the ion 2c

by the consecutive losses of molecules of ethynol and

acetylene, followed by the loss of a hydrogen radical.

To verify the validity of our fragmentation routes obtained

during the CID-MS/MS analysis, we exchanged the labile

hydrogen atoms from the corresponding two OH groups

with the deuterium atom of CH3OD prior to injection into the

ESI source. We expected to obtain at least three different

deuteriated molecules, which had incorporated one to three

deuterium atoms. We postulated that the addition of the

third deuterium atom can be attributed to enolization or to

the protonation of the nitrogen atom.

As expected, in the ESI mass spectrum of the deuterated

naloxone hydrochloride 2, we noticed the incorporation of

three deuterium atoms, accounting for the main ions at m/z

329.1563, 330.1596, and 331.1652 (see Fig. 2(C)).

Second-generation product ion spectra of the selected

singly charged precursor monomer deuteriated ions at m/z

329.1563, 330.1596, and 331.1652 were also recorded;

however, these spectra are not shown here. By carefully

studying these product ion spectra it was possible to confirm

the majority of the proposed structures of the product

ions described in Scheme 5. As discussed earlier in the case

of naloxonazine dihydrochloride 1, it is essential to point


DOI: 10.1002/rcm

A

C

B

400380360340320300280260240220200180160140120100m/z

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

12.0

13.0

14.0

282.12

240.08254.11

226.08198.08161.05 212.06184.06 253.08264.09153.06128.05115.05 280.10

400380360340320300280260240220200180160140120100m/z

0

5

10

15

20

25

30

35

40

45

50

55

60 268.11

161.05226.07

212.05

240.10213.07199.06 253.09

120.07 184.05 225.07165.06115.05 153.06147.03 238.07 266.09187.06 205.08 242.10

400380360340320300280260240220200180160140120100m/z

0

100

200

300

400

500

600

700

800

212.05

253.09184.06156.07128.05

Figure 6. Third-generation product ion scans of the selected intermediate ions obtained from the singly charged

[MþH–HCl]þ ion of naloxone hydrochloride 2 (A) at m/z 282.14, (B) at m/z 268.13, and (C) at m/z 253.10.


out that the masses of the diagnostic product ions have

increased with the expected number of deuterium atoms, thus

confirming the genesis of formation of all the product ions.

Precusor ion scans of some selected ions ofnaloxone hydrochloride 2The precursor ion scans of the product ions at m/z 310,

268, 253, 240, 227 and 212 were also recorded with a


quadrupole-hexapole-quadrupole MS/MS instrument, to

confirm the origins of their formation and to ascertain their

CID-MS/MS genesis.

The precursor ion scan of the ion at m/z 310 indicated that

it originated from the singly charged protonated mole-

cule [MþH–HCl]þ. The precursor ion scan of the ion

[MþH–HCl–H2O–C3H6]þ at m/z 268 showed that it was

formed from the ions at m/z 310 and 328. The precursor ion


DOI: 10.1002/rcm

1074 N. Joly et al.

scan of the [MþH–HCl–H2O–C3H6]þ ion at m/z 253 showed

that it originated from either of the ions at m/z 310 or 328. The

precursor ion scan of the ion at m/z 240 showed that it was

formed from the ions at m/z 268, 282, 310, and 328. The

precursor ion scan of the ion at m/z 227 showed its multiple

genesis, originating from the ions atm/z 240, 254, 268, 282, 310

and 328. Finally, the precursor ion scan of the ion at m/z 212

showed that it originated from the ions at m/z 254, 253, 268,

282, 310 and 328. These precursor ion scan analyses have

confirmed the origins of formation of the major diagnostic

product ions and ascertained their CID-MS/MS genesis.

CONCLUSIONS

Mass spectral analysis of the zwitteronic salts naloxonazine

dihydrochloride 1 and naloxone hydrochloride 2, which

are common morphine opiate receptor antagonists, has

been facilitated by electrospray ionization tandem mass

spectrometry (ESI-MS/MS) using a QqToF-MS/MS hybrid

instrument. An abundant singly charged ion [MþH–2HCl]þ

at m/z 651.3170 and the doubly charged ion [Mþ2H–2HCl]2þ

atm/z 326.1700 were found for naloxonazine dihydrochloride

1; while the singly charged ion [MþH–HCl]þ at m/z 328.1541

was observed for naloxone hydrochloride 2. Collisio-

n-induced dissociation in the atmospheric/vacuum inter-

phase was promoted by using a higher declustering potential

and this permitted the recording of CID-MS/MS spectra of

the intermediate product ions.

During this study we have noted the formation of the

radical ions 1c, 1d, 1f and 1g, at m/z 592.22, 574.22, 532.19 and

405.18, respectively, obtained during the product ion scan of

the singly charged dimeric ion [MþH–2HCl]þ 1a at m/z

651.26 for naloxonazine hydrochloride.

Similarly, we propose that the ion 2e0 at m/z 212.07 was a

b-distonic ion, formed during the product ion scan of the

singly charged precursor ion [MþH–HCl–H2O–C3H6]þ 2c at

m/z 253.10 by the loss of 41 Da assigned as a loss of ethynol

and the addition of a hydrogen radical.

Finally, the use of low-energy CID-MS/MS permitted

the rationalization of the exact fragmentation pathways.

Furthermore, product and precursor ion scans of the selected


intermediate ions allowed rationalization of the fragmenta-

tion patterns of these zwitteronic salts.

AcknowledgementsJoseph Banoub acknowledges the financial support of the

Natural Sciences and Engineering Research Council of

Canada for a Discovery Grant and Applied Biosystems-MDS

SCIEX for generously providing extra ionization sources

necessary for ESI-QqToF-MS experiments.

REFERENCES

1. Millan MJ. Trends Pharmacol. Sci. 1990; 11: 70.2. Baker AK, Meert TF. J. Pharmacol. Exp. Ther. 2002; 302: 1253.3. Bale RN, Van Stone WW, Kuldau JM, Engelsing TMJ, Elash-

off RM, Zarcone VP. Arch. Gen. Psychiatry 1980; 37: 179.4. Rapaka RS, Porreca F. Pharm. Res. 1991; 8: 1.5. Crain SM, Shen KF. Proc. Natl. Acad. Sci. USA 1995; 92: 10540.6. McQuay H. Lancet 1999; 353: 2229.7. Spector SJ. J. Pharmacol. Ther. 1971; 178: 253.8. Hand CW, Moore RA, McQuay HJ, Allen MC, Sear JW. Ann.

Clin. Biochem. 1987; 14: 153.9. Tyrefors N, Hyllbrant B, Ekman L, Johansson M, Langstrom

B. J. Chromatogr. A 1996; 729: 279.10. Blanchet M, Bru G, Guerret M, Bromet-Lepetit M, Bromet N.

J. Chromatogr. A 1999; 854: 93.11. Zheng M, McErlane KM, Ong MC. J. Pharm. Biomed. Anal.

1998; 16: 971.12. Joly N, El Aneed A, Martin P, Cecchelli R, Banoub J. Rapid

Commun. Mass Spectrom. 2005; 19: 3119.13. Crain SM, Shen KF. Pain 2000; 84: 121.14. Mhatre M, Holloway F. Alcohol 2003; 29: 109.15. Di Donna L, Mazzotti F, Sindona G, Tagarelli A. Rapid

Commun Mass Spectrom. 2005; 19: 1575.16. Cordaro M, Di Donna L, Grassi G, Maiuolo L, Mazzotti F,

Perri E, Sindona G, Tagarelli A. Eur. J. Mass Spectrom. 2004;10: 691.

17. Di Donna L, Maiuolo L, Mazzotti F, De Luca D, Sindona G.Anal Chem. 2004; 76: 5104.

18. Gentil E, Lesimple A, Le Bigot Y, Delmas M, Banoub J. RapidCommun. Mass Spectrom. 1994; 8: 869.

19. Chalmers MJ, Hakansson K, Johnson R, Smith R, Shen JW,Emmet MR, Marshall AG. Proteomics 2004; 4: 970.

20. Cuyckens F, Rozenberg R, de Hoffman E, Claeys M. J. MassSpectrom. 2001; 36: 1203.

21. Cole RB. J. Mass Spectrom. 2000; 35: 763.22. Holmes JL. Org. Mass Spectrom. 1985; 20: 169.23. Yates BF, Bouma WJ, Radom L. J. Am. Chem. Soc. 1984; 106:

5805.24. Eberlin MN. J. Mass Spectrom. 2006; 41: 14.25. Tomazela DM, Sabino AA, Sparrapan R, Gozzo FC, Eberlin

MN. J. Am. Soc. Mass Spectrom. 2006; 17: 1014.


DOI: 10.1002/rcm

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