Identification of isobaric product ions in electrospray ionization mass spectra of fentanyl using multistage mass spectrometry and deuterium labeling

Article abstract of DOI:10.1002/rcm.4673

Isobaric product ions cannot be differentiated by exact mass determinations, although in some cases deuterium labeling can provide useful structural information for identifying isobaric ions. Proposed fragmentation pathways of fentanyl were investigated by electrospray ionization ion trap mass spectrometry coupled with deuterium labeling experiments and spectra of regiospecific deuterium labeled analogs. The major product ion of fentanyl under tandem mass spectrometry (MS/MS) conditions (m/z 188) was accounted for by a neutral loss of N-phenylpropanamide. 1-(2-Phenylethyl)- 1,2,3,6-tetrahydropyridine (1) was proposed as the structure of the product ion. However, further fragmentation (MS³) of the fentanyl m/z 188 ion gave product ions that were different from the product ion in the MS/MS fragmentation of synthesized 1, suggesting that the m/z 188 product ion from fentanyl includes an isobaric structure different from the structure of 1. MS/MS fragmentation of fentanyl in deuterium oxide moved one of the isobars to 1Da higher mass, and left the other isobar unchanged in mass. Multistage mass spectral data from deuterium-labeled proposed isobaric structures provided support for two fragmentation pathways. The results illustrate the utility of multistage mass spectrometry and deuterium labeling in structural assignment of isobaric product ions.

Full text of DOI:10.1002/rcm.4673

RAPID COMMUNICATIONS IN MASS SPECTROMETRY
Rapid Commun. Mass Spectrom. 2010; 24: 2547–2553
Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.4673
Identification of isobaric product ions in electrospray
ionization mass spectra of fentanyl using multistage
mass spectrometry and deuterium labeling
Wisut Wichitnithad1, Terence J. McManus2 and Patrick S. Callery3
*
1Department of Pharmaceutical Technology Program, Chulalongkorn University, Bangkok 10330, Thailand
2Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, WV 26506, USA
3Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, WV 26506, USA
Received 18 May 2010; Revised 18 June 2010; Accepted 21 June 2010
Isobaric product ions cannot be differentiated by exact mass determinations, although in some cases
deuterium labeling can provide useful structural information for identifying isobaric ions. Proposed
fragmentation pathways of fentanyl were investigated by electrospray ionization ion trap mass
spectrometry coupled with deuterium labeling experiments and spectra of regiospecific deuterium
labeled analogs. The major product ion of fentanyl under tandem mass spectrometry (MS/MS)
conditions (m/z 188) was accounted for by a neutral loss of N-phenylpropanamide. 1-(2-Phenylethyl)-
1,2,3,6-tetrahydropyridine (1) was proposed as the structure of the product ion. However, further
fragmentation (MS3) of the fentanyl m/z 188 ion gave product ions that were different from the
product ion in the MS/MS fragmentation of synthesized 1, suggesting that the m/z 188 product ion
from fentanyl includes an isobaric structure different from the structure of 1. MS/MS fragmentation
of fentanyl in deuterium oxide moved one of the isobars to 1 Da higher mass, and left the other isobar
unchanged in mass. Multistage mass spectral data from deuterium-labeled proposed isobaric
structures provided support for two fragmentation pathways. The results illustrate the utility of
multistage mass spectrometry and deuterium labeling in structural assignment of isobaric product
ions. Copyright # 2010 John Wiley & Sons, Ltd.
Multistage mass spectrometry and labeling with deuterium
provide useful approaches to the elucidation of chemical
structures and quantification. In quantitative mass spec-
trometry, it is desirable to have product ions of reproducible
intensity.1 Isobaric and isomeric fragment ions sharing the
same exact mass but not sharing the same internal energy or
fragmentation pathway could vary in intensity depending
on instrument operating parameters. Variations in intensity
of product ion signals could compromise accuracy and
precision of quantitative measurements. Awareness of the
presence of isobaric fragment ions can guide the choice
of ions to monitor in quantitative analytical method
development.
exchange depending on the characteristics of the cation
detected. As an example, two isobaric mass spectral ions, one
with a cationic charge on carbon and the second carrying a
charge derived from a protonated heteroatom, have by
definition the same mass. Replacement with deuterium of the
proton attached to the heteroatom increases the mass by 1 Da
for each deuterium atom incorporated into the molecule,
whereas the carbon-based cation may not exchange and
maintains the same mass. Thus, deuterium labeling provides
a means for separating these isobaric ions in preparation for
further fragmentation stages. Fentanyl is an example of a
compound that exhibits mass spectral isobaric fragment ions.
Fentanyl (Fig. 1) is a potent narcotic analgesic used widely
for the treatment of pain, and subject to illicit use.3 Several
quantitative liquid chromatography/tandem mass spectrom-
etry (LC/MS/MS) methods for measuring fentanyl have been
published based on monitoring a [m/z 337 ! m/z 188]
transition.4–7 An ion at m/z 188 is also observed in the electron
ionization (ESI) spectrum as a fragment ion as well as in the
matrix-assisted laser desorption/ionization (MALDI)-MS/
MS spectrum of fentanyl.5,8 A structure of the major product
ion (m/z 188) in the mass spectrum of fentanyl has been
proposed that is consistent with a neutral loss of N-
phenylpropanamide.4–9 Interestingly, flash pyrolysis of fen-
tanyl yields the same neutral loss of N-phenylpropanamide
resulting in the formation of 1-(2-phenylethyl)-1,2,3,6-tetra-
hydropyridine (1) (Fig. 1) as a major product.10 The ESI mass
One approach to differentiating isobaric ions involves
deuterium labeling coupled with multistage mass spectrom-
etry experiments. Compounds characterized as [M þ H]þ
2
ions are shifted to [M þ H]þ ions in the presence of
deuterated protic solvents, such as deuterium oxide.
Hydrogen/deuterium (H/D) exchange has proved to be a
simple and rapid approach to gaining evidence supporting
the identification of unknown structures and H/D exchange
is useful in identifying drug metabolites.2 Isobaric com-
pounds can be differentiated in some cases by deuterium
*Correspondence to: P. S. Callery, Department of Basic Pharma-
ceutical Sciences, West Virginia University, Morgantown, WV
26506-9530, USA.
E-mail: pcallery@hsc.wvu.edu
Copyright # 2010 John Wiley & Sons, Ltd.

2548 W. Wichitnithad, T. J. McManus and P. S. Callery
Figure 2. Synthesis of regioselectively deuterium-labeled
analogs of 1-(2-phenylethyl)-1,2,3,6-tetrahydropyridine (1).
Figure 1. Structures of fentanyl and 1-(2-phenylethyl)-
1,2,3,6-tetrahydropyridine (1).
yield compound 1 (273 mg, 80.1%). 1H-NMR (CDCl3): d
(ppm) ¼ 7.4–7.2 (m, 5H, ArH); 5.9–5.8 (m, 1H, vinylic H); 5.8–
5.7 (m, 1H, vinylic H); 3.2–3.1 (d, 2H, C-6 H); 2.9–2.8 (m, 2H,
N-CH2); 2.8–2.6 (m, 4H, C-2 H and CH2Ar); 2.3–2.2 (bs, 2H,
C-3 H).
spectrum of 1 ([MH]þ, m/z 188) has the same mass as the
[fentanyl ! m/z 188] transition.
In the course of our studies on the breakdown products
derived from smoking fentanyl, we synthesized 1 and then
compared the MS/MS product ion spectrum of 1 with the
MS3 spectrum obtained from fragmentation of the MS/MS
product ion (m/z 188) from fentanyl. The resulting spectra
were different, suggesting that the identity of the
[fentanyl ! m/z 188] ion has a structure other than that of
the thermal degradation product, compound 1.
[6-2H]-1-(2-Phenylethyl)-1,2,3,6-tetrahydro-
pyridine (1-d1)
By adapting the method of Kalgutkar and Castagnoli,15
compound 1 (200 mg, 1.07 mmol) was oxidized to the
intermediate N-oxide with 3-chloroperbenzoic acid (77%
m-CPBA) (239 mg, 1.39 mmol) at 08C for 2 h. The product was
purified by chromatography on basic alumina eluted with
CH2Cl2/MeOH (90:10) to yield the N-oxide of 1 (187 mg,
86.1%). Without further purification, the N-oxide of 1 in
CH2Cl2 was then converted into the corresponding dihy-
dropyridinium compound by using trifluoroacetic anhy-
dride (TFAA) (966 mg, 4.60 mmol) under a nitrogen atmos-
phere at 08C for 1 h. The solvent was removed under reduced
pressure and the residue was subsequently reduced with
NaBD4 (116 mg, 2.76 mmol) in methanol under a nitrogen
atmosphere at 08C for 4 h. Volatile solvents were removed
and the residue was extracted with ethyl acetate (3 ꢀ 15 mL).
The combined organic layers were dried (Na2SO4). The crude
product was purified under the same condition as 1 to yield
1-d1 (151 mg, 75.1%). 1H-NMR (CDCl3): d (ppm) ¼ 7.4–7.2 (m,
5H, ArH); 5.9–5.8 (m, 1H, vinylic H); 5.8–5.7 (m, 1H, vinylic
H); 3.2–3.1 (m, 1H, C-6 H); 3.0–2.9 (m, 2H, N-CH2); 2.8–2.6
(m, 4H, C-2 H and CH2Ar); 2.3–2.2 (bs, 2H, C-3 H). ESI-MS
To assign structures to the m/z 188 ion, studies were done
using deuterium-labeled solvents, regiospecific deuterium
labeling, and multistage mass spectrometry in an ion trap
mass spectrometer.
EXPERIMENTAL
Chemistry
Deuterium oxide, NaBD4, perdeuteropyridine, and all other
reagents were supplied by Aldrich Chemical Company (St.
Louis, MO, USA). The syntheses of 1 and deuterium-labeled
analogs of 1 were based on modified procedures developed
for the synthesis of substituted 1,2,3,6-tetrahydropyri-
dines.11–16 A general synthetic scheme is outlined in Fig. 2
1-(2-Phenylethyl)-1,2,3,6-tetrahydropyridine (1)
Briefly, pyridine (200 mg, 2.53 mmol) and 2-chloroethylben-
zene (336 mg, 2.53 mmol) were condensed at 1408C to yield 1-
(2-phenylethyl)pyridinium chloride (2) (425 mg, 76.5%)
following the method of Cui et al.14 Compound 2 (400 mg,
1.82 mmol) was reduced with NaBH4 (207 mg, 5.47 mmol) in
methanol at 08C for 4 h. After removal of volatile solvents
under reduced pressure, the residue was extracted with ethyl
acetate (3 ꢀ 15 mL). The combined organic layers were dried
(Na2SO4) and filtered. The crude product was purified by
chromatography on basic alumina eluted with CH2Cl2 to
2
m/z 189 ([MHþ], 85.5% H).
[2,6-2H2]-1-(2-Phenylethyl)-1,2,3,6-tetrahydro-
pyridine (1-d2)
Substitution of NaBD4 (229 mg, 5.46 mmol) for NaBH4 in the
synthesis of 1 provided 1-d2 (269 mg, 78.1%) following an
adapted method of Mabic et al.16 1H-NMR (CDCl3): d
(ppm) ¼ 7.4–7.2 (m, 5H, ArH); 5.9–5.8 (m, 1H, vinylic H);
5.8–5.7 (m, 1H, vinylic H); 3.2–3.0 (m, 1H, C-6 H); 3.0–2.9
Copyright # 2010 John Wiley & Sons, Ltd.
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DOI: 10.1002/rcm

Isobaric product ions in ESI mass spectra of fentanyl 2549
(m, 2H, N-CH2); 2.8–2.6 (m, 3H, C-2 H and CH2Ar); 2.3–2.2
(90:10) to yield the 1-(2-phenylethyl)piperidine N-oxide
(754 mg, 3.67 mmol, 86.9%), which was then treated with
TFAA (386 mg, 18.3 mmol) in CH2Cl2 under a nitrogen
atmosphere at 08C to yield 4 (812 mg, 73.4%). 1H-NMR
(CDCl3): d (ppm) ¼ 8.5–8.4 (d, 1H, C-6 H); 7.4–7.0 (m, 5H,
ArH); 4.2–4.1 (t, 2H, N-CH2); 3.7–3.6 (m, 2H, C-2 H); 3.3–3.2
(t, 2H, CH2Ar); 2.8–2.7 (m, 2H, C-5 H); 2.0–1.9 (m, 2H, C-3 H);
1.8–1.6 (m, 2H, C-4 H).
2
(bs, 2H, C-3 H). ESI-MS m/z 190 ([MHþ], 90.9% H2).
[2,3,4,5,6-2H5]-1-(2-Phenylethyl)-1,2,3,6-tetrahydro-
pyridine (1-d5)
Pentadeutero-labeled 1 (1-d5) was prepared by substituting
[2,3,4,5,6-2H5]-pyridine (200 mg, 2.38 mmol) for pyridine in
the synthesis of 1 to yield 2-d5 (430 mg, 80.5%). Reduction of
2-d5 (400 mg, 1.78 mmol) with NaBH4 (207 mg, 5.47 mmol) in
1
methanol at 08C gave compound 1-d5 (265 mg, 77.4%). H-
Mass spectrometry
NMR (CDCl3): d (ppm) ¼ 7.4–7.2 (m, 5H, ArH); 3.2–3.1 (d, 1H,
C-6 H); 3.0–2.9 (m, 2H, N-CH2); 2.8–2.6 (m, 3H, C-2 H and
CH2Ar); 2.3–2.2 (bs, 1H, C-3 H). ESI-MS m/z 193 ([MHþ],
ESI mass spectra were recorded on an ion trap mass
spectrometer (LCQ DECA, Thermo Scientific, San Jose, CA,
USA). Sample inlet was by direct infusion from a syringe
pump. The electrospray needle voltage was 5.2 kV, the
heated capillary voltage was 13 V, and the capillary
temperature was maintained at 2008C. Sheath gas and
auxiliary flow rates were set at 20 arbitrary units. The sample
flow rate was approximately 5 mL/min. The normalized
collision energy was set at 45%, activation time was 30 ms,
and the isolation width was 0.8 Da for multistage mass
spectra. The samples were dissolved either in 0.1% formic
acid in methanol, or in deuterium oxide. [MH]þ ions were
converted into [MD]þ ions when compounds were dissolved
in 0.1% formic acid in deuterium oxide (>99.5% 2H2).
Compound 3 and 3-d1 under these conditions exchanged the
aniline N-H with deuterium.
2
87.0% H5).
N-(1-(2-Phenylethyl)-4-piperidinyl)aniline (3)
Hydrolysis of fentanyl HCl (100 mg, 0.30 mmol) was
accomplished by heating fentanyl solutions in 4 N HCl at
1008C for 72 h. The residue was then neutralized by using 5 N
NH4OH and extracting with ethyl acetate to provide
compound
3
(42 mg, 55.9%). 1H-NMR (CDCl3):
d
(ppm) ¼ 7.4–7.2 (m, 7H, Ar H); 6.8–6.7 (t, 1H, ArH); 6.7–6.6
(d, 2H,ArH); 3.7–3.5 (bs, 1H); 3.5–3.3 (m, 1H); 3.1–2.9 (m, 2H);
2.9–2.8 (m, 2H, N-CH2); 2.7–2.6 (m, 2H, CH2Ar); 2.4–2.2 (m,
2H); 2.2–2.1 (m, 2H); 1.6–1.5 (m, 2H).
[2-2H]- N-(1-(2-Phenylethyl)-4-piperidinyl)-
aniline (3-d1)
A triple quadrupole instrument (Quatro Micro, Waters
Corp., Milford, MA, USA) was used to record spectra of
collision-generated ion fragments of the fentanyl [MH]þ ion.
Samples of fentanyl dissolved in deuterium oxide
were directly infused using a syringe pump at a flow rate
of 10 mL/min. Capillary voltage was set at 3.50 kV with a
cone voltage of 38.0 V. Desolvation gas flow rate and cone gas
flow rate were maintained at 400 and 50 L/h, respectively.
Desolvation temperature was set at 3008C. Collision energy
was 24 (arbitrary units). Collision gas pressure was set at
3.0 ꢀ 10ꢁ3 mbar.
By adapting the method of Kalgutkar and Castagnoli,15
fentanyl free base (100 mg, 0.30 mmol) was converted into the
corresponding fentanyl N-oxide intermediate with 3-chloro-
perbenzoic acid (77% m-CPBA) (67 mg, 0.30 mmol) at 08C for
1 h, and the product purified by chromatography on basic
alumina eluted with CH2Cl2/MeOH (90:10) to yield fentanyl
N-oxide (97 mg, 92.6%). Subsequently, TFAA (289 mg,
1.38 mmol) was added dropwise to fentanyl N-oxide in
CH2Cl2 under a nitrogen atmosphere at 08C to yield the
3,4,5,6-tetrahydropyridinium analog of fentanyl. Without
purification, reduction of the crude intermediate with NaBD4
(46.1 mg, 1.10 mmol) in methanol at 08C gave [2-2H]-fentanyl
(fentanyl-d1) (75 mg, 80.8%). Following the method for the
preparation of 3, acid hydrolysis of fentanyl-d1 (50 mg,
0.15 mmol) was accomplished by heating fentanyl solutions
in 4 N HCl at 1008C for 72 h and purified by silica column
chromatography using 1% triethylamine in ethyl acetate/
hexane (50:50) to yield 3-d1 (38 mg, 91.3%). 1H-NMR (CDCl3):
d (ppm) ¼ 7.4–7.2 (m, 7H, ArH); 6.8–6.7 (t, 1H, ArH); 6.7–6.6
(d, 2H, ArH); 3.6–3.5 (bs, 1H, N-H), 3.5–3.3 (m, 1H); 3.1–2.9
(m, 1H); 2.9–2.8 (m, 2H, N-CH2); 2.7–2.6 (m, 2H, CH2Ar); 2.4–
2.2 (m, 2H); 2.2–2.1 (m, 2H); 1.6–1.5 (m, 2H). ESI-MS m/z 282
RESULTS AND DISCUSSION
Investigation of isobaric product ions of
fentanyl
The precursor ion and major MS/MS product ions of
fentanyl in methanol are shown in Table 1. The most
prominent ion at m/z 188 can be accounted for by a neutral
loss of N-phenylpropanamide. Multistage fragmentation of
m/z 188 (MS3) gives a product ion of m/z 105 assigned as a
phenylethyl cation, along with several other ions including
m/z 132, 134 and 146 (Fig. 3(a)).
The spectrum of fentanyl in deuterium oxide indicated
2
([MHþ], 86.2% H).
replacement of one hydrogen for deuterium giving a
2
[M þ H]þ ion at m/z 338 containing one deuterium atom.
1-(2-Phenylethyl)-2,3,4,5-tetrahydropyridinium
TFA (4)
MS/MS fragmentation of m/z 338 yielded m/z 189 as a
product ion, consistent with deuterium incorporation, and
m/z 188 in which deuterium had not been incorporated. The
intensity of the m/z 188 ion was approximately of equal
abundance to the m/z 189 ion. If scrambling of the deuterium
label had occurred during the MS/MS process, the ions in the
MS3 spectrum of m/z 188 and 189 would have been identical
other than the presence of deuterium. This was not the case.
1-(2-Phenylethyl)piperidine (800 mg, 4.23 mmol), prepared
by reductive amination of phenylacetaldehyde (784 mg,
5.87 mmol) with piperidine (500 mg, 5.87 mmol) and NaBH4
(667 mg, 17.6 mmol), was converted into the N-oxide
intermediate. The crude product was purified by chroma-
tography on basic alumina eluted with CH2Cl2/MeOH
Copyright # 2010 John Wiley & Sons, Ltd.
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2550 W. Wichitnithad, T. J. McManus and P. S. Callery
Table 1. Precursor and major product ions of fentanyl ana-
ESI conditions in a triple quadrupole mass spectrometer
showed only the m/z 188 ion and no m/z 189 ion, indicating
that isobaric product ion formation may differ among
instrument or ionization types.
logs
m/z
Precursor ion
Compound
[M þ H]þ
Product ions
Regioselective deuterium labeling
Deuterium incorporation in specific locations on the piper-
idine ring of fentanyl and fentanyl fragments and multistage
mass spectrometry provided evidence in support of the
proposed fragmentation pathways. Labeled compounds
fentanyl-d1, 1-d1, 1-d2, 1-d5, and hydrolyzed fentanyl-d1 were
prepared. A listing of major product ions in the mass spectra
of labeled compounds is shown in Table 1.
Fentanyl
Fentanyl-d1
1
1-d1
337
338
188
189
190
193
281
281, 216, 188, 105
282, 217, 189, 188, 105
134, 105
134, 105
135, 105
1-d2
1-d5
135, 105
188, 146, 134, 105
Despropionyl
fentanyl (3)
Despropionyl
fentanyl-d1 (3-d1)
4
282
188
188, 146, 147, 134, 135, 105
146, 132, 105
Proposed fragmentation pathways of isobaric
ions
Two pathways (A and B) are proposed that can reasonably
explain the occurrence of isobaric product ions in the
multistage mass spectrum of fentanyl. Deuterium labeling
was used to separate the isobaric m/z 188 ions by converting
one isobaric ion into m/z 189 by replacement of one hydrogen
with deuterium and leaving the other isobaric ion (m/z 188)
unchanged (Fig. 5).
The MS3 spectrum of m/z 189 showed a major product ion of
m/z 135 (Fig. 3(b)), whereas the MS3 spectrum of m/z 188 gave
m/z 146 and 132 as major product ions (Fig. 3(c)). It is clear
that product ions m/z 188 and 189 do not share the same
structure, other than differing by one deuterium atom. The
conclusion drawn from this observation is that in the mass
spectrum of fentanyl, the MS/MS m/z 188 ion consists of at
least two isobaric species. It is interesting to note that the
MS/MS spectrum of deuterium-exchanged fentanyl under
Collisional activation of fentanyl causes the elimination of
N-phenylpropanamide as a neutral loss, most likely as a
result of collisional heating. The resulting MS/MS ion
detected in the fentanyl spectrum is the same mass as the
protonated olefin 1 observed at m/z 188. Thermal decompo-
sition of fentanyl hydrochloride is a model of this reaction, as
reported by Manral et al.10
Pathway A
Assignment of structure 1 to the fentanyl m/z 188 product ion
is consistent with pathway A. When the protonation source is
deuterium oxide, the product ion m/z 188 will become m/z 189
in a H/D exchange process. The MS3 dissociation pattern of
transition [m/z 189 ! m/z 135] follows the same pathway as the
MS/MS dissociation of compound 1 in deuterium oxide
(Fig. 5(a)).
Pathway A represents retention of deuterium in the MS/
2
MS dissociation of the [M þ H]þ ion generated from fentanyl
in deuterium oxide, consistent with protonation at the most
probable site on the fentanyl molecule. The protonated
piperidine nitrogen with a pKa of about 817 is the most likely
Figure 3. (a) Multistage fragmentation of fentanyl in water
[MH]þ ! m/z 188 ! m/z 105, 132, 134, and 146; (b) MS3
spectrum of the deuterated product ion (m/z 189); and (c)
MS3 spectrum of isobaric (not deuterated) product ion (m/z
188) from fentanyl in deuterium oxide.
Figure 4. Proposed fragmentation pathways of fentanyl by
ESI-MS/MS.
Copyright # 2010 John Wiley & Sons, Ltd.
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Isobaric product ions in ESI mass spectra of fentanyl 2551
Figure 5. Proposed mechanism of pathway A. (a) Elimination of N-phenylpropanamide from fen-
tantyl in water or deuterium oxide by a six-centered rearrangement. (b) Elimination of N-phenylpro-
panamide from fentantyl-d1 in deuterium oxide. (c) Multistage fragmentation of despropionyl fentanyl
and fentanyl-d1 in deuterium oxide. (d) Proposed RDA ring cleavage of deuterium isotopomers of 1.
Copyright # 2010 John Wiley & Sons, Ltd.
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DOI: 10.1002/rcm

2552 W. Wichitnithad, T. J. McManus and P. S. Callery
site of cation formation of fentanyl. Elimination of N-
Figure 5(c) shows the proposed structures of despropionyl
fentanyl mass spectrum in deuterium oxide following
pathway A. Acid hydrolysis of the propionyl group from
fentanyl provided fentanyl derivatives no longer capable of
the six-centered reaction proposed for the formation of 1
from fentanyl, either thermally or after ionization within the
mass spectrometer. The mass spectra of despropionyl
fentanyl and despropionyl fentanyl-d1 in deuterium oxide
show neither the m/z 135 nor the m/z 136 ion in the MS3 of m/z
188 from the transition [m/z 283 ! m/z 188] and the MS3 of
m/z 189 from transition [m/z 284 ! m/z 189], respectively,
which supports the proposed participation of the amide
carbonyl in a rearrangement reaction.
2
phenylpropanamide from [M þ H]þ in
a six-centered
rearrangement involving abstraction of a proton from C-3
of the piperidine ring provides a product ion that retains the
deuterated piperidine nitrogen. The MS3 transition [m/z
338 ! m/z 189 ! m/z 135] is reasonably explained by a retro-
Diels-Alder (RDA) rearrangement and a neutral loss of
butadiene from m/z 189 (Fig. 5(a)).
Fentanyl labeled with one deuterium atom specifically on
the carbon adjacent to the piperidine nitrogen was prepared
as a tracer that would be potentially useful in verifying a
RDA ring-opening fragmentation process. [2-2H]-Fentanyl
was synthesized by oxidizing fentanyl to an N-oxide
intermediate followed by treatment with trifluoroacetic acid
to give the 2,3,4,5-tetrahydropyridinium intermediate.
Reduction of the intermediate with NaBD4 introduced one
deuterium at C-2 of fentanyl and produced [2-2H]-fentanyl.
The MS/MS spectrum of [2-2H]-fentanyl in deuterium oxide
showed the transition of [2-2H-fentanyl, m/z 339 ! m/z
190 ! m/z 135 and 136], which is consistent with a RDA
rearrangement proposed in pathway A (Fig. 5(b)).
Evidence in support of pathway A was provided by
analysis of the mass spectral characteristics of 1 regiospe-
cifically labeled with deuterium including 1-d1 (labeled on
the allylic position, C-6), 1-d2 (labeled with one deuterium
atom at each C-2 and C-6), and 1-d5 (deuterated at carbons 2,
3, 4, 5, and 6). Figure 5(d) shows the proposed RDA
fragmentation of deuterium isotopomers of 1 that provides
analogous ions to those ions observed in the MS3 spectrum of
Figure 6. Proposed mechanism of pathway B. (a) Multistage fragmentation of fentanyl with 4 as an
intermediate. (b) Multistage fragmentation of fentanyl-d1 in water and deuterium oxide showing
transitions [m/z 338 ! 189 ! 147] and [m/z 338 ! 189 ! 146] explained by a hydride (m/z 147) or
deuteride (m/z 146) rearrangement.
Copyright # 2010 John Wiley & Sons, Ltd.
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Isobaric product ions in ESI mass spectra of fentanyl 2553
hydrogen or deuterium did not rearrange, only m/z 147
would have been observed.
In comparison with pathway A, pathway B shows
a
relatively more prominent multistage transition of
[m/z 338 ! m/z 188 ! m/z 105] representing the neutral loss
of azabicyclo[2.2.0]hexane or 2,3,4,5-tetrahydropyridine in
the third stage. In pathway B, either the N-stabilized C-4
carbocation or iminium ion 4 provides neutral losses
accounting for an intense phenylethyl cation (m/z 105)
(Figs. 4 and 6(a)).
Figure 7. MS spectrum of 1-(2-phenylethyl)-2,3,4,5-tetrahy-
dropyridium ion (4, m/z 188) and MS/MS product ions from
m/z 188.
Multistage mass spectral analysis of selectively deuterated
fentanyl and fentanyl analogs provides support for a
proposed mechanism of fentanyl fragmentation and the
characterization of an unusual observation of an isobaric
fragment ion.
2
fentanyl-d1 in deuterium oxide, [M þ H]þ, m/z 339 ! m/z
190 ! m/z 135 and 136.
Acknowledgements
Financial support from the Thailand Research Fund
through the Royal Golden Jubilee PhD Program (Grant
No. PHD/0217/2548) to W. Wichitnithad is acknowledged.
The triple quadrupole data were generated in the Clinical
Pharmacology Core Facility of the Mary Babb Randolph
Cancer Center, West Virginia University, Morgantown,
WV, USA.
Pathway B
Pathway B is consistent with either protonation on the amide
functionality, or rearrangement of a proton from the
piperidinium nitrogen to the amido group. Although from
solution acid-base first principles amide protonation is a
considerably less likely site of protonation of fentanyl, upon
fragmentation the resulting secondary carbocation can be
stabilized through space by the piperidine nitrogen unshared
pair of electrons. The major product ion in pathway B does
not contain a proton or deuteron from solvent (Fig. 4). Thus,
pathway B shows only m/z 188 in the MS/MS dissociation of
REFERENCES
1. Kaufmann A, Butcher P, Maden K, Widmer M, Giles K, Uria
D. Rapid Commun. Mass Spectrom. 2009; 23: 985.
2. Prakash C, Shaffer CL, Nedderman A. Mass Spectrom. Rev.
2007; 26: 340.
3. Wong SC, Mundy L, Drake R, Curtis JA, Wingert WE. J. Med.
Toxicol. 2010; 6: 9.
deuterium-exchanged fentanyl [M þ H]þ ion, indicating
2
that deuterium has not been incorporated into this product
ion. MS3 dissociation of m/z 188 is different from the MS/MS
spectrum of tetrahydropyridine 1.
4. Koch DE, Isaza R, Carpenter JW, Hunter RP. J. Pharm.
Biomed. Anal. 2004; 34: 577.
5. Pihlainen K, Grigoras K, Franssila S, Ketola R, Kotiaho T,
Kostiainen R. J. Mass Spectrom. 2005; 40: 539.
6. Lurie IS, Iio R. J. Chromatogr. A 2009; 1216: 1515.
7. Takashina Y, Naito T, Mino Y, Kagawa Y, Kawakami J.
J. Clin. Pharm. Ther. 2009; 34: 523.
8. Cheng MT, Kruppa GH, McLafferty FW. Anal. Chem. 1982;
54: 2204.
9. Peer CJ, Clay DJ, Glover HL, Renninger KL, Kraner JC,
Callery PS. J. Anal. Toxicol. 2008; 32: 709.
10. Manral L, Gupta PK, Suryanarayana MVS, Ganesan K,
Malhotra RC. J. Thermal Anal. Calorimetry 2009; 96: 531.
11. Kalgutkar AS, Castagnoli K, Hall A, Castagnoli N Jr. J. Med.
Chem. 1994; 37: 944.
12. Sugasawa S. Berichte der Ceutschen Chemischen Gesellschaft
1941; 74B: 459.
13. Efange SMN, Michelson RH, Remmel RP, Boudreau RJ,
Dutta AK, Freshler A. J. Med. Chem. 1990; 33: 3133.
14. Cui L, Sapagovas V, Lattermann G. Liquid Crystals 2002; 29:
1121.
15. Kalgutkar AS, Castagnoli N Jr. J. Med. Chem. 1992; 35: 4165.
16. Mabic S, Rimoldi JM, Castagnoli N Jr. J. Label. Compd.
Radiopharm. 1997; 39: 409.
In pathway B, allylic rearrangement of a C-4 carbocation to
become a more stable cation at C-6 with a concomitant 1,3-
hydride shift is reasonably indicated. The resulting 2,3,4,5-
tetrahydropyridinium compound 4 was considered as a
possible source of the [m/z 337 ! m/z 188] MS/MS transition
from fentanyl (Fig. 6(a)). Iminium 4 was synthesized as a
potential model for this rearrangement ion. MS/MS of 4
showed the [m/z 188 ! m/z 146, 132, and 105] transition
(Fig. 7) which was also observed in the MS3 spectrum of
fentanyl [m/z 337 ! m/z 188 ! m/z 146, 132, and 105],
suggesting that a rearrangement to the stable degenerate
cation 4 is a reasonable postulation.
Fentanyl-d1 labeled with one deuterium at C-2 was
prepared as a marker for a potential hydride rearrangement
in the formation of 4. The [fentanyl-d1 ! m/z 189 ! m/z 146,
147] transition is explained by the loss of a hydrogen or
deuterium from the C-2 ring position (Fig. 6(b)). The m/z 147/
146 intensity ratio was calculated to be 0.29. If the C-2
17. Thurlkill RL, Cross DA, Scholtz JM, Pace CN. J. Cardiothorac.
Vasc. Anesth. 2005; 19: 759.
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 2547–2553
DOI: 10.1002/rcm