Structural determination of the novel fragmentation routes of morphine opiate receptor antagonists...

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2005; 19: 3119–3130

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.2179

Structural determination of the novel fragmentation

routes of morphine opiate receptor antagonists using

electrospray ionization quadrupole time-of-flight

tandem mass spectrometry

Nicolas Joly1, Anas El Aneed2, Patrick Martin1, Romeo Cecchelli1 and Joseph Banoub2,3*1Laboratoire de le Barriere Hemato-Encephalique, E.A. 2465, Departement de Chimie, Universite d’Artois, Bethune, France2Department of Biochemistry, 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, Canada

Received 29 June 2005; Revised 1 September 2005; Accepted 1 September 2005

Electrospray ionization quadrupole time-of-flight (ESI-QqToF) mass spectra of naltrindole hydro-

chloride 1, naltriben mesylate 2, and naltrexone hydrochloride 3, a common series of morphine opi-

ate receptor antagonists, were recorded using different declustering potentials. Low-energy

collision-induced dissociation (CID) MS/MS experiments established the fragmentation routes of

these compounds. In addition, re-confirmation of the various established fragmentation routes was

effected by conducting a series of ESI-CID-QqTof-MS/MS experiments using non-conventional

quasi MSn (up to MS8) product ion scans, which were initiated by CID in the atmospheric pres-

sure/vacuum interface using a higher declustering potential. Precursor ion scan analyses were

also performed with a conventional quadrupole-hexapole-quadrupole tandem mass spectrometer

and allowed the confirmation of the genesis of some diagnostic ions. Copyright # 2005 John Wiley

& Sons, Ltd.

The opiates are a naturally occurring basic alkaloid series of

compounds, such asmorphine andheroin,which have a high

pharmacological activity, and lead to a tolerance phenomen-

on and familiarization causing ‘drug addiction’.1

Opiates induce sleep, relieve pain, and cause sedation and

pleasure by acting on the brain’s Mu, Delta and Kappa

peptide neurotransmitter receptors releasing endorphins

and encephalins. The opiates release an excess of dopamine

in the brain resulting in a constant need for opiate, to block the

opiod receptor. The abuse of opiates and the subsequent

development of dependence has become a major health

problem in many parts of the world,2 leading to the

development of several compounds to treat this depen-

dence.3 These compounds are antagonists which act through

one or more of the opiate receptors responsible for the

narcotic activities associated with drugs such as morphine

and heroin.4 Three examples of such compounds are

naltrindole hydrochloride5 1, naltriben mesylate6 2, and

naltrexone hydrochloride7 3 (also called narcan).

These compounds are effective in treating drug addiction;

however, there is a high failure rate due to non-compliance

followed by relapse which is often due to the heightened

effects of heroin following two or three days without the

antagonist. This is often seen as a reward by the addict and

thus the treatment often results in further heroin use and

sometimes overdosing due to greater drug tolerance follow-

ing treatment.7

The detection and positive identification of the opiate

antagonist, especially in the presence of opiates, is of great

importance in monitoring compliance or abuse by treatment

subjects. Also with some of the lower doses now being

studied and recommended to avoid relapse, highly sensitive

and specific methods are required for the detection and

monitoring of levels of these compounds in biological

samples. This may be useful in a clinical or forensic situation.

A number of reports in the literature have shown high-

performance liquid chromatography/electrospray ioniza-

tion tandem mass spectrometry (HPLC/ESI-MS/MS) to be

successful in the identification of morphine and morphine

metabolites in plasma.8,9 Gas chromatography/negative

chemical ionization mass spectrometry has also been used

to identify morphine in plasma samples.10

Recently, capillary electrophoresis/ESI quadrupole-ion

trap tandem mass spectrometry has been used to identify

morphine, codeine and their metabolites in urine samples.11

As a continuation of our interest in the MS and MS/MS of

bioactive molecules,12–14 we now report on the structural

characterization of a series of salts of naltrexone hydro-

chloride 1, naltrinben mesylate 2, and naltrindole hydro-

chloride 3 (see Scheme 1).

To our knowledge there have been no previous studies on

the mass spectrometric characterization of these types of

Copyright # 2005 John Wiley & Sons, Ltd.

*Correspondence to: J. Banoub, Department of Biochemistry, Mem-orial University of Newfoundland, St John’s, Newfoundland,A1B 3V6, Canada.E-mail: [email protected]/grant sponsor: Natural Sciences and EngineeringResearch Council of Canada.

morphine opiate receptor antagonists. The salts of the

morphine antagonists 1–3 make them excellent candidates

for the ESI process. Evidence of the possible fragmentation

routes was first obtained by collision-induced dissociation

(CID) in the atmospheric pressure/vacuum interface using

various higher declustering potentials.15 Structural confir-

mation was also obtained from low-energy CID-MS/MS

analysis of diagnostic fragment ions derived from the

protonated precursor molecules. Rationalization of the

fragmentation routes was achieved by obtaining the product

ion and precursor ion spectra of the various intermediate

ions.16,17

EXPERIMENTAL

Sample preparationThe derivatives naltrindole hydrochloride 1, naltriben mesy-

late 2, and naltrexone hydrochloride 3were purchased from

Tocris Cookson (St. Louis, MO, USA). The molecular struc-

tures of these derivatives are shown in Scheme 1.

ESI mass spectrometryMass spectrometry was performed using an Applied Biosys-

tems API QSTAR XL MS/MS quadrupole orthogonal time-

of-flight (QqToF)-MS/MS hybrid instrument capable of ana-

lyzing 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. A total of 0.1mg of each morphine antagonist was

dissolved in methanol/water (10:1) to achieve a concentra-

tion of 0.1 mmol/mL. Aliquots (3 mL) were infused into the

mass spectrometer with an integrated Harvard syringe

pump at a rate of 1mL/min. Product ion spectra were

obtained arising from fragmentation in the radio-frequency

(RF)-only quadrupole equipped with a LINAC (linear accel-

eration pulsar high pressure) collision cell of the QqToF-MS/

MShybrid instrument.Nitrogenwas used as the collision gas

forMS/MS analyses with collision energies varying between

10 and 35 eV. Collision energy (CE) and CID gas conditions

were adjusted such that the precursor ion remained abun-

dant.

Precursor ion scan 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. Precursor ion scans of mass-

selected ions were obtained by collision of these ions with

argon in the (RF-only) hexapole. A personal computer

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

service pack 3) equipped with Masslynx 3.3 Mass Spectro-

metry 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 naltrindolehydrochloride 1, naltriben mesylate 2, andnaltrexone hydrochloride 3The ESImass spectrum (positive ionmode) of the naltrindole

hydrochloride 1 was recorded with a declustering potential

(DP) of 30V and gave a monocharged [MþH–HCl]þ ion at

m/z 415.2028 (Fig. 1(A)). This ion was identical to the

[MþH]þ ion of naltrindole obtained from naltrindole hydro-

chloride 1 by neutralization with sodium hydroxide at

pH> 9, and acidification with formic acid.

Similarly, the ESI mass spectrum of naltriben mesylate 2

was recorded with DP 30V (Fig. 1(B)). In this spectrum we

noted the formation of the [MþH–CH3SO3H]þ ion at m/z

416.1872 and the sodiated species [MþNa–CH3SO3H]þ at

m/z 438.1752.

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

fragmentation of both naltrindole hydrochloride 1 and

naltriben mesylate 2 (data not shown) and resulted in the

formation of a series of diagnostic fragment ions which were

also formed during the CID-MS/MS analysis. The genesis of

these ions and their proposed structures are discussed and

presented in the CID-MS/MS analysis sections.

The ESI-MS of naltrexone hydrochloride 3 recorded with

DP 30V resulted in the formation of the [MþH–HCl]þ ion at

m/z 342.1734, in addition to an adductwhichwas formedwith

the methanol mobile phase, namely the [MþH–

HClþMeOH]þ ion at m/z 374.2081 (Fig. 1(C)). With

DP¼ 100V we noticed the formation of the [MþH–HCl]þ

ion at m/z 342.1734, the sodiated adduct [MþNa–HCl]þ at

m/z 364.1607, and the methanol adduct at m/z 374.2081. We

also noted the formation of an abundant ion at m/z 324.1606,

which was assigned as the [MþH–HCl–H2O]þ ion formed

by the loss ofwater fromm/z 342.1734.Minor fragment ions at

m/z 282.1452, 270.1142 and 267.1285 were also noted.

ESI-QqToF-MS/MS of natrindole hydrochloride 1Low-energy CID-MS/MS analyses were conducted to ratio-

nalize the pathways leading to the various fragmentations

Scheme 1. Representation of the molecular structures of naltrindole hydrochloride 1, naltriben mesylate 2, and

naltrexone hydrochloride 3.

Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 3119–3130

3120 N. Joly et al.

observed in the conventional mass spectra obtainedwith dif-

ferent declustering potentials. The product ion scan of the ion

at m/z 415.2215 afforded a series of product ions (see

Fig. 2(A)), the formation of which is presented in Scheme 2.

The m/z values of these product ions are in agreement with

those obtained by CID in the atmospheric pressure/vacuum

interface generated with a higher declustering potential of

100V. In this rationale, the product ion scan of m/z 415.2215

afforded m/z 397.2101 by the straightforward loss of water.

The ion at m/z 343.1621 was formed by the loss of 1,3-

butadiene fromm/z 397.2101. The ion atm/z 300.1161waspro-

duced by elimination of a molecule ofN-(cyclopropylmethy-

lene)aziridine (97Da) from m/z 397.2101. The ion at m/z

326.1349 was formed by elimination of 1-cyclopropylmethy-

lamine (71Da) fromm/z 397.2101. It is interesting to note that

the minor ion at m/z 314.1320 was formed from m/z 397.2101

by the loss of the N-(cyclopropylmethylene)methylamine

molecule (83Da). The ion at m/z 308.1207 was formed by a

straightforward elimination of water from m/z 326.1349.

The ion at m/z 282.1031 was rationalized as being formed by

two separate processes. It could be formed fromm/z 300.1161

by the elimination ofwater, or by elimination of amolecule of

acetylene andamolecule ofwater fromm/z 326.1349. Thepro-

duct ion atm/z 280.1260was formed fromm/z 308.1207 by the

loss of ethylene. Finally, we noted that the ion atm/z 254.1079

was formed from m/z 282.1031 by the opening of the furan

Figure 1. ESI-Tof-MS scans obtained at DP¼ 30V for naltrindole hydrochloride 1 (A), naltriben mesylate

2 (B), and naltrexone hydrochloride 3 (C).

ESI-CID-MS/MS of morphine opiate receptor antagonists 3121


ring followed by tautomerization into the keto derivative,

with subsequent elimination of CO and ring contraction

(see Scheme 2).

Please note that Scheme 2 is comprised of the overall

fragmentationpatterns deduced in this experiment and in the

following sections, illustrating the various product and

precursor ion scan experiments for naltrindole hydrochlor-

ide 1.

Also, to avoid confusion, it is crucial to comprehend that

throughout this manuscript, and, in the following proposed

schemes, the ion masses that have been underlined corre-

spond to the mass of the selected precursor ions used for the

additional CID-MS/MS experiments. In addition, themasses

of the resulting product ions originate from the last precursor

scan analysis.

ESI-QqToF-MSn of selected precursor ionsobtained from naltrindole hydrochloride 1A second-generation product ion scan (also called quasiMS3)

of the selected precursor fragment ion at m/z 397.2132

afforded, inter alia, a series of product ions at m/z 343.1624,

326.1360, 308.1218, 300.1169, 282.1037, 280.1245, 254.1065,

226.0930, 212.0787, and 130.0667 (data not shown). In this

new CID-MS/MS analysis the new product ion at m/z

Figure 2. Product ion scan (CID-MS/MS) of the precursor ion atm/z 415.2215 (A), the quasiMS4 product

ion scan for m/z 343.1656 (C), and the quasi MS5 product ion scan for m/z 308.1233, obtained from the

naltrindole hydrochloride molecule 1.

3122 N. Joly et al.


Scheme 2. Proposed overall fragmentation pathways obtained from the various product and precursor ion scans

and quasi MSn experiments of naltrindole hydrochloride 1.



130.0667was assigned as being formedby the fission ofC-14–

C-8 and C-5–C-6.

Another second-generation product ion scan experiment

was performed on the fragment ion at m/z 343.1656 and

afforded a series of product ions, as shown in Fig. 2(B). The

ion atm/z 212.0787 is formed fromm/z 343.1624 by the loss of a

molecule ofmethyl indole (131Da). The ion atm/z at 194.1074

arose from the loss of water from m/z 212.0829 (Fig. 2(B)).

Please observe that it has been established that the selected

ion at m/z 343.1656, used for the previous CID-MS/MS

experiment, was also formed from the precursor ion at m/z

397.2132. Therefore, perhapswe canpostulate that in fact, this

is a third-generation product ion scan or quasi MS4 rather

than quasiMS3. In the following rationale, we shall adopt this

postulate to describe further the following MSn analysis.

Hence, a fourth-generationproduct ion scanor quasiMS5of

the ion atm/z 326.1380 afforded, inter alia, a series of product

ions at 308.1239, 307.1150, 291.1190, 290.1115, 280.1256, and

278.1084 (data not shown) (Scheme 2). Simple losses of a

hydrogen radical, ammonia and a hydrogen molecule were

observed. These losses and the corresponding structures of

the elimination product ions are presented in Scheme 2.

The product radical ion at m/z 307.1150 was formed from

m/z 308.1239, by the loss of a hydrogen radical (not shown in

Scheme 2).

Please note that the formation of radical ions in this

rationale is not a new finding as, in 1994, we were the first

authors to report the formation of a radical ion from a

positively charged even-electron product ion obtained

during the ESI-CID-MS/MS scans 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

A fifth-generation product ion scan or quasiMS6 of the ion

at m/z 308.1239 afforded, inter alia, a series of product ions

(shown in Fig. 2(C)). Theunassignedproduct radical ion atm/

z 279.1177was tentatively proposed as being formed from the

ion at m/z 280.1262, by the loss of a hydrogen radical.

A sixth-generation product ion scan or quasi MS7 of the

intermediate fragment ion atm/z 300.1186 afforded, inter alia,

the product ions atm/z 285.0919, 282.1062, 272.1201, 257.0095,

254.1072, and 244.1121 (data not shown). The product radical

ion at m/z 285.0919 was obtained from m/z 300.1186 by

opening of the furan ring followed by reduction (oxygen

radical expulsion and hydrogen transfer). The ion at m/z

272.1201 was generated fromm/z 300.1186, which arose from

the opening of the C-4–O bond of the furan ring, followed by

tautomerization into the keto form, and the loss of CO, and

subsequent ring contraction. The ion at m/z 244.1121 was

formed fromm/z 272.1201, by tautomerization of the C-3-OH

group, and the same process as was discussed previously

(Fig. 2(C) and Scheme 2).

Finally, a seventh-generation product ion scan or quasiMS8

of the fragment ion at m/z 282.1108 afforded the major

product ion atm/z 254.1074 and theminor ions atm/z 253.0997

and226.0888 (data not shown). The radical ion atm/z 253.0987

was formed from m/z 254.1074 by the loss of a hydrogen

radical (Scheme 2). For the sake of brevity in this rationale,we

have only presented the product ion spectra of the ions atm/z

415.2215 (MS2), 343.1656 (MS4) and307.1156 (MS6) (Fig. 2).All

the proposed structures of the product ions obtained in the

quasiMSn experiments are tentatively assigned in Scheme 2.

Figure 3. Precursor ion scans of the ions at m/z 300 (A), 282 (B), 254 (C), and130 (D) obtained from the

naltrindole hydrochloride molecule 1.

3124 N. Joly et al.


Precursor ion scans of some selected ionsof naltrindole hydrochloride 1The precursor ion scans of the fragment ions at m/z 300, 282,

254 and 130 were also recorded with a quadrupole-hexapole-

quadrupoleMS/MS instrument. The precursor ion scan of the

m/z 300 ion indicated that it was formed from eitherm/z 397 or

the protonated molecule at m/z 415 (Fig. 3(A)). The precursor

ion scanofm/z 282 indicated that itwas formed fromanyof the

ions atm/z 300, 326, 397 and415 (Fig. 3(B)). In addition, thepre-

cursor ion scan of the fragment ion at m/z 254 showed that it

originated from any of the ions at m/z 282, 300, 326, 397, and

415 (Fig. 3(C)). Finally, the precursor ion scan of the fragment

ion atm/z 130 showed that it originated from eitherm/z 397 or

the protonatedmolecule atm/z 415 (Fig. 3(D)). The structure of

this product ion is indicated in Scheme 2.

It is logical to deduce that themultiple origins of this series

of product ions, obtained by the various CID MS/MS

experiments and precursor ion scans, arose by either

concerted or consecutive multiple neutral losses. In this

context, note that ‘concerted’ or ‘consecutive’ losses of the

manyproduct andprecursor ions in theMS/MS experiments

simply means that the molecules are both lost at the same

time, andwithin the same reaction region, the collision cell, of

the QqToF hybrid tandem mass spectrometer.

Figure 4. Product ion scan (CID-MS/MS) of the precursor ion atm/z 416.2074 (A), the quasiMS3 product

ion scan for m/z 398.1837 (B), and the quasi MS5 product ion scan for m/z 301.1006 (C), obtained from

the naltriben mesylate molecule 2.



ESI-QqToF-MS/MS analysis of naltribenmesylate 2To reveal the correct fragmentation routes of the [MþH–

CH3SO3H]þ ion, the product ion spectrum of m/z 416.2074

was recorded, and afforded, inter alia, a series of major

product ions, shown in Fig. 4(A), which were tentatively

rationalized and presented in Scheme 3. The ion at m/z

398.1943 was formed by the elimination of water from

the C-12 hydroxyl group located in the cis-position to the

N-(cyclopropylmethylene)ethylenamine bridgehead of the

molecule. The ion at m/z 344.1469 appeared to be formed

by two different processes. It can be formed either from the

concerted or consecutive elimination of water and butadiene.

We also noted the formation of the ion atm/z 301.0981, which


and quasi MSn experiments of naltriben mesylate 2.

3126 N. Joly et al.


arose either by the loss of amolecule ofN-(cyclopropylmethy-

lene)vinylamine (97Da) fromm/z 398.1943 or by the loss of an

aziridine molecule (43Da) fromm/z 344.1469.

ESI-QqToF-MSn analysis of selected precursorions obtained from naltriben mesylate 2Second-generation MS/MS experiments, or quasi MS3, were

generated by selecting the precursor ion at m/z 398.1837, to

produce, inter alia, a series of product ions as shown in

Fig. 4(B). The t ion at m/z 356.1323 was formed from the pre-

cursor ion by a retro-Diels-Alder (RDA) reactionwith the loss

of a molecule of C2H2O (42Da) and the consecutive opening

of the C-11–C-12 covalent (aromatic) bond to afford the di-

acetylinic open-chain ion, which recombines by a 1,4-

cycloaddition (see Scheme 3). The ion at m/z 327.1160 was

formed by the elimination of a molecule of 1-cyclopropyl-

methylamine (71Da) from m/z 398.1943. It should be noted

that the ion at m/z 301.0891 was formed from m/z 344.1356

by the loss of a molecule of aziridine (43Da). This ion was

also formed in the conventional CID-MS/MSof the precursor

[MþH–CH3SO3H]þ ion at m/z 416.2074. The ion at m/z

255.0912 was formed from m/z 398.1837 by the consecutive

or concerted losses of molecules of C6H11N, CO and water

(97Da).

Figure 5. Product ion scan (CID-MS/MS) of the precursor ion at m/z 342. 1846 (A), the quasi MS3

product ion scan form/z 324.1601 (B), and the quasiMS5 product ion scan form/z 267.1396 (C), obtained

from the naltrexone hydrochloride molecule 3.



A third-generation MS/MS experiment (quasi MS4) was

effected on the selected precursor ion at m/z 344.1478 which

gave, inter alia, the product ions at m/z 309.1221, 301.1054,

273.1073, and 245.1134 (data not shown). In this quasi MS4

product ion scan we noticed the formation of three new

product ions at m/z 309.1221, 273.1073 and 245.1134. The ion

at m/z 309.1221 was produced from m/z 344.1478 by the

consecutive losses of molecules of water and ammonia. m/z

301.1054 afforded the product ion at m/z 273.1073 by the loss

of 28Da which occurred by the opening of the furan ring,

followed by tautomerization into the keto form and subse-

quent loss of CO. The product ion at m/z 245.1134 appears to


and quasi MSn experiments of naltrexone hydrochloride 3.

3128 N. Joly et al.


be formed fromm/z 273.1073 following the tautomerizationof

the C-3 hydroxyl group into the keto form, followed by the

loss of CO and ring contraction.

A fourth-generation product ion scan or quasi MS5 of the

selected precursor ion at m/z 301.1006 afforded, inter alia, the

series of product ions shown in Fig. 4(C).

The product ions observed in this quasi MS5 experiment

were generated by similar mechanisms to the one discussed

earlier for naltrindole hydrochloride 1 and included losses of

H2O, H2, H and acetylene species (RDA reaction, refer to

Scheme 3 for details). The ion at m/z 286.0748 was generated

from m/z 301.1006 by the expulsion of an atom of oxygen

followed by a hydrogen transfer from a neutral molecule.

Finally, the quasi MS6 of the precursor ion at m/z 255.0939

affordedasoleproduct ionatm/z226.0910,whichwasdeduced

as being formed by the concerted losses initiated by a RDA

fragmentation (loss of a molecule of acetylene), the loss of a

hydrogenmolecule and the loss of a hydrogen radical (29Da).

Please note that, in this quasi MS6 experiment, the ion at m/z

226.0910,generated fromtheprecursor ionatm/z255.0939,was

also present as a product ion in the quasi MS3 ion scan of the

precursor ion at m/z 398.1837, and in the quasi MS5 product

ion scan of the precursor ion atm/z 301.1006 (Scheme 3).

This series of product ion scans have allowed us to verify

the various novel fragmentation routes of the morphine

antagonist naltriben mesylate 2.

ESI-QqToF-MS and ESI-QqToF-MS/MS analysesof naltrexone hydrochloride 3The product ion spectrum of the [MþH–HCl]þ ion at m/z

342.1846 gave, inter alia, a major series of product ions (see

Fig. 5(A)). The ion atm/z 324.1656was formed by elimination

of water from m/z 342.1846 (see Scheme 4). The ion at m/z

282.1538 was deduced as being formed by a RDA fragmenta-

tion, with release of a molecule of ethynol (C2H2O) as pre-

viously discussed. The ion at m/z 270.1246 was formed by

elimination of a molecule of butadiene from m/z 324.1656.

The radical ion atm/z 267.1387was formedby the consecutive

losses of a cyclopropylmethylene radical (55Da) and ahydro-

gen molecule from m/z 324.1656. In addition, the ion at m/z

267.1387 canoriginate fromm/z 270.1245 by consecutive elim-

ination of a molecule of hydrogen and a hydrogen radical.

The ion atm/z 228.1124 was initiated by the RDA fragmenta-

tion of m/z 270.1245 involving the loss of a molecule of ethy-

nol. The ion atm/z 227.0906was attributed to the formation of

a radical ion formed fromm/z 270.1245, by the elimination of

ethynol, followed by an additional loss of a hydrogen radical

(Scheme 4). As expected, this morphine analogue 3 fragmen-

ted in a manner very similar to naltrindole hydrochloride 1

and naltriben mesylate 2. This analogy in the fragmentation

patterns is anticipated due to their structural similarities (see

Schemes 1 and 2).

ESI-QqToF-MSn analysis of selected precursorions produced from naltrexone 3Second-generation product ion spectra, or quasi MS3, were

produced by selecting the ion at m/z 324.1601, to yield, inter

alia, the ions shown in Fig. 5(B). The genesis of the majority

of the product ions has already been discussed except for

m/z 226.0865, which was formed from m/z 270.1156 by a con-

secutive RDAelimination of amolecule of ethynol and loss of

a hydrogen molecule.

A third-generation product ion scan was produced by a

quasi MS4 experiment by selecting the precursor ion at m/z

Figure 6. Precursor ion scans of the ions at m/z 282 (A), 270 (B), 267 (C), and 227 (D) obtained from the

naltrexone hydrochloride molecule 3.



282.1552 to afford, inter alia, the ions atm/z 280.1486, 267.1403,

254.1363, 228.1133, 227.1050, 226.0995, 213.0972, and 212.1020

(data not shown). The rationale leading to these product ions

is self-explanatory and is presented in Scheme 4, which

resembles, to a large extent, the fragmentation pathways

observed for the CID-MS/MS analysis of naltrindole hydro-

chloride 1 and naltriben mesylate 2, presented in Schemes 2

and 3.

The fourth-generation product ion spectrum, or quasiMS5,

of the selected precursor ion at m/z 267.1396 gave a series of

product ions (seeFig. 5(C). The radical ion atm/z 239.1283was

generated from m/z 267.1396 by the loss of CO. Finally, the

radical ion atm/z 212.0806 originates fromm/z 267.1396 by the

consecutive losses of molecules of HCN and CO.

Precursor ion scans of selected ions obtainedfrom naltrexone hydrochloride 3Figure 6 shows thee precursor ion scans of the fragment ions

atm/z 282, 270, 267, and 227. Themultiple origins of these ions

confirm, once again, the consecutive, stepwise genesis of their

precursor ions, as deduced from the series of product/pre-

cursor ion scans depicted in Scheme 4.

CONCLUSIONS

The aim of this communication is to define the novel ESI-MS

fragmentation routes of this common series of morphine opi-

ate receptor antagonists, naltrindole hydrochloride 1, naltri-

ben mesylate 2, and naltrexone hydrochloride 3, by

electrospray ionization. Low-energy CID-MS/MS product

ion and precursor ion scan experiments provided character-

istic fingerprint ionswhich helped in the establishment of the

fragmentation routes. In addition, we have rationalized the

formation of each individual precursor and product ion by

performing quasi MSn (up to MS8) experiments initiated by

CID in the atmospheric pressure/vacuum interface using a

higher declustering potential. This study will be used by

the French Blood Brain Barrier Laboratory in the actual clin-

ical identification of the metabolites in plasma from subjects

treated with this series of antagonists. We have prepared a

new series of b-D-gluco-uronopyranoside derivatives of this

series ofmorphine antagonistswith non-labelled anddeuter-

ated analogues of the synthetic b-D-gluco-uronides. The

syntheses and the ESI-CID-QqToF-MS/MS of this series of

novel b-D-gluco-uronides will be reported when completed.

In conclusion, we can reaffirm that low-energy CID-MS/

MS analysis is an excellent tool to confirm,without doubt, the

novel fragmentation routes and the genesis of the ions which

have been tentatively assigned in this manuscript.

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.

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