Study of complexes of C2- and C6-dihydroceramides with transition metal ions using electrospray ionization tandem mass spectrometry (ESI-MS/MS)

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Complexes of C2- and C6-dihydroceramides with Transition Metal Ions Bull. Korean Chem. Soc. 2009, Vol. 30, No. 2 397
Study of Complexes of C2- and C6-dihydroceramides with Transition Metal Ions
Using Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS)
Jin-Yi Lim, Avvaru Praveen Kumar, Changdae Kim,† Chuljin Ahn, Young-Jae Yoo, and Yong-Ill Lee*
Department of Chemistry, Changwon National University, Changwon 641-773, Korea. *E-mail: yilee@changwon.ac.kr
†Department of Physics, Mokpo National University, Mokpo 534-729, Korea
Received March 21, 2008, Accepted December 26, 2008
The complexes of C2- and C6-dihydroceramides with transition metal ions have been investigated by using
Electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The formation and fragmentation pathways of
several doubly charged cluster ions as well as singly charged cluster ions of C2- and C6-dihydroceramides with
transition metal ions have studied by ESI-MS/MS in the positive mode. Under ESI conditions, dihydroceramides
form singly and doubly charged complexes with transition metal ions (Mn2+, Fe2+, Co2+, Ni2+, and Zn2+ except
Cu2+) with the compositions of [DHCer+M+2H2O-H]+, [2DHCer+M+2H2O-H]+, [3DHCer+M+2H2O-H]+, [2DHCer
+M]2+, [3DHCer+M]2+, [4DHCer+M]2+, [5DHCer+M]2+, and [6DHCer+M]2+ (DHCer = C2- or C6-dihydroceramide,
M = transition metal ion). The different complexation behavior of copper is responsible for relatively lower
affinity of dihydroceramides to copper compared to those of other transition metals. It is also found that in the mass
spectrum of the dihydroceramide complexes with copper(II), [2DHCer+Cu-H]+ was observed with considerable
intensity as well as [2DHCer+Cu+2H2O-H]+ due to its different geometry from those of other metals.
Key Words: Transition metal complexes, C2-dihydroceramide, C6-dihydroceramide, Electrospray ioniza-
tion tandem mass spectrometry
Introduction
Ceramides are a family of lipid molecules, found in high
concentrations within the cell membrane of cells. Struc-
turally, the ceramides exhibit a long aminoalcoholic chain
covalently bound via an amide linkage to a fatty acyl moiety
that can vary in length (synthetic short chain ceramides: C2-,
C4-, C6-, . . . or natural ceramides: C16-, C18-, C20-, . . .).1 They
are one of the component lipids that make up sphingomyelin,
one of the major lipids in the lipid bilayer. Ceramides have
recently been identified as key signal molecules which mediate
many biological functions such as cell growth, differentiation,
senescence, and apoptosis.2-7 Cell permeable analogs of
ceramide, such as C2-ceramide and C6-ceramide, have been
used to analyze the role of ceramide in intracellular signaling.8
Apoptotic activities of C2 ceramide and C2 dihydroceramide
against HL-60 cells are examined because the circumstances
around the primary hydroxyl group is important for the
apoptotic activity.9,10 The wide range biological effects of
ceramides that depend on cell type, receptors involved,
sub-cellular location, and concentration suggest the existence
of several downstream targets for distinct intracellular path-
ways.
Transition metal ions are known to be important in
biological, environmental and many other chemical systems.
Transition metal complexes are exploited to promote organic
synthesis and transition metal cations, especially multiply
charged cations, have a pivotal role in enzymatic processes in
biological systems.11 In most papers, the authors describe
complexes of monovalent cations with various organic ligands;
however, bivalent cations are also a subject of interest. The
ESI mass spectrometry is recognized almost immediately
after the introduction of this technique and a significant
number of papers on this subject have already been published.
The first information on the ESI spectra of [3M+Met]2+
complex ions is published in 1990 by Katta et al.12 The ESI
mass spectrometry has a potentiality for studying weak,
non-covalent interactions between biomolecules and metal
cations. Structural characterization of ceramides in positive or
negative mode has been reported by electrospray ionization
tandem mass spectrometry (ESI- MS/MS)13-15 and also by fast
atom bombardment tandem mass spectrometry (FAB-MS/
MS).16,17 Kerwin et al.18 investigated sphingomyelins and
fragmentation studies of ceramides in existence of lithium ion
have demonstrated by ESI-MS/MS.19 The analysis of ceramides
with high sensitivity and selectivity without prior separation
and derivatization has been studied by using ESI-MS.20,21
From the literature survey, it can be realize that tandem
mass spectrometry is a useful tool for the study of formation
and fragmentation pathways of biological metal complexes
due to MSn (multiple-stage tandem mass spectrometry).
Fortunately, there are no previous and extensive studies on
ceramide complexation with transition metal ions, so we
aimed to explore the complexes of ceramides with transition
metal ions. In our previous study,22 we reported the complexes
of C2-ceramide with transition metal cations using ESI-
MS/MS, now we wish to report the transition metal com-
plexes of C2- and C6-dihydroceramides, similarities and dif-
ferences in complexation between the C2- and C6-dihydro-
ceramides using ESI-MS/MS in the positive mode.
Experimental
Materials and reagents. D-erythro-N-Acetyl sphinganine
(C2-dihydroceramide), N-hexanoyldihydro-sphingosine (C6-
dihydroceramide), manganese(II) chloride, ferrous(II) chloride,

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398 Bull. Korean Chem. Soc. 2009, Vol. 30, No. 2Jin-Yi Lim et al.
HO
HN
O
OH

HO
HN
O
OH
Figure 1. Structures of D-erythro-N-Acetylsphinganine (C2-dihy-
droceramide) (a); N-Hexanoyl dihydrosphingosine (C6-dihydroce-
ramide) (b)
F
A : [ DHCer(2) + H] +
B : [ 2DHCer(2) + Mn] 2+
C : [ DHCer(2) + Mn + 2H2O – H ] +
D : [ 3DHCer(2) + Mn ] 2+
E : [ 4DHCer(2) + Mn ] 2+
F : [ 2DHCer (2)+ Mn + 2H2O – H ] +
G: [ 5DHCer (2)+ Mn ] 2+
H : [ 6DHCer(2) + Mn ] 2+
I : [ 3DHCer(2) + Mn + 2H2O – H ] +
776.4
714.2
1119.2
885.4
542.7
433.3
1057.1
371.1
344.2
A
B
C
E
G
H
I
D
m/z
m/z
R
elative A
bundance (%
)
300400500600 700800900 10001100 12001300
0
20
40
60
80
100
Relative Abundance (%)
Figure 2. ESI positive ion mass spectrum of aqueous methanol
(25/75%) solution containing a mixture of C2-dihydroceramide
(5.0x10-5M) dissolved in methanol and manganese(II) chloride
(5.0x10-4 M) dissolved in aqueous methanol(50/50%) at 20V tube
lens offset voltage.
cobalt(II) chloride, nickel(II) chloride, copper(II) chloride,
and zinc(II) chloride were purchased from Sigma Chemical
Co. (St.Louis, MO, USA). Methanol of gradient grade (Merck,
Darmstadt, Germany) was used for mass spectrometry. Other
chemicals and solvents were purchased from Aldrich
Chemical Co. (St. Louis, MO, USA) and used without further
purification.
The metal complex solutions of C2- and C6-dihydrocer-
amides were prepared by mixing the aqueous methanol
solutions of transition metal chlorides at concentrations of 1.0
× 10-3 M to 1.0 × 10-5 M and 1.0 × 10-4 M to 10-6 M of ceramide
solution in methanol with different concentration ratios of
transition metal ion and ceramide (1:1 to 1:200) just before the
infusion into the mass spectrometer. The samples thus prepared
were infused in aqueous methanol (25/75 %) solutions using a
syringe pump at a flow rate of 5 µl min-1 and the corresponding
ions were introduced into the mass spectrometer by electro-
spray.
Mass spectrometry. All experiments were performed by
using an LCQ-Advantage ion trap mass spectrometer (Thermo
Finnigan Co., San Jose, CA, USA) equipped with an ESI
source. The sample solution was infused into the elec-
trospray interface by a syringe pump at a flow rate of 5 µL
min-1. Operation conditions were as follows; spray voltage,
4.5 kV; capillary voltage, 3 V; heated capillary temperature,
200 oC; and sheath gash (N2), 20 arb. Helium gas admitted
directly into the ion trap was used as the buffer gas to improve
trapping efficiency and as the collision gas for CID experi-
ments. Tube lens offset voltages were set by using a tune file
created by auto tuning of the LCQ on the ion signal of interest
if not specified. CID experiments were performed by setting
the isolation width between 5 and 10 mass units depending on
the species of focus and the activation amplitude at 5 - 25% of
5 V peak-to-peak in resonance excitation RF voltage. All
mass spectra recorded were the average of 20 consecutive
scans.
Results and Discussion
The structures of C2- and C6-dihydroceramides were shown
in Fig. 1, (a) and (b), respectively. The complexation of C2-
and C6-dihydroceramides with transition metal ions was
studied by ESI-MS/MS in the positive mode. Fig. 2 is the full-
mass spectrum of the complexes of C2-dihydroceramide with
manganese(II) ion. The spectrum includes several complex
ions, not only singly charged cluster ions but also doubly
charged cluster ions like [nDHCer(2)+Mn]2+ (n : ranging from
2 to 6).
Fragmentation of complexes of C2-dihydroceramide with
Mn2+. Prior to MSn of [5DHCer(2)+Mn]2+, we performed MS/
MS and MSn for a variety of ions ranging from [3DHCer(2)+
Mn+2H2O–H]+ to [6DHCer(2)+Mn]2+. Among them, MSn (n
= 2~5) spectra for one of the doubly charged ions, [5DHCer-
(2)+Mn]2+ was illustrated in Fig. 3 (Here, 2 in parenthesis
indicates the carbon number of fatty acid). The MS/MS of
[5DH Cer(2)+Mn]2+ (m/z 885) gave rise to [4DHCer(2)+Mn]2+
ion at m/z 714 by the loss of one C2-dihydroceramide. The
MS3 of [4DHCer(2)+Mn]2+ which was the outstanding ion
produced by MS2 of [5DHCer(2)+Mn]2+ yields fragment ion
at m/z 543 by the lack of one C2-dihydroceramide. By MS4 of
precursor ion at m/z 543, there were several product ions such
as [DHCer(2)+H]+ at m/z 344, [2DHCer(2)+Mn]2+ at m/z
371, [2DHCer(2)+Mn–H–H2O]+ at m/z 722, and [2DHCer
(2)+Mn–H]+ at m/z 740. The important thing to be noticed is
that the intensity of [2DHCer+Mn+2H2O-H]+ was outstan-
dingly fierce by comparison with that of [4Cer+M]2+ or
[5Cer+M]2+ from C2-ceramide.22 This difference is made by
the fact that a double bond of C2-ceramide can enhance the
possibility of hydrogen bonding and therefore, four or five
C2-ceramides can provide sufficient stability to manganese
ion. That was, the difference in sphingoid backbone of ceramide
has a substantial influence on the stability of complex ions.
The MS5 spectrum of [2DHCer(2)+Mn]2+ (m/z 371) included
two singly charged cluster ions [DHCer(2)+H–H2O]+ at m/z 326
and [DHCer(2)+Mn–H]+ at m/z 397. [DHCer(2)+H–H2O]+
was generated by protonated C2-dihydroceramide with lack of
one H2O and reduction of one C2-dihydroceramide, one man-
ganese, and one H2O from [2DHCer(2)+Mn]2+. While [DHCer(2)
+Mn-H]+ was formed by the loss of one C2-dihydroceramide
and hydrogen ion. The different CID energies with 0.81V,
(a)
(b)

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Complexes of C2- and C6-dihydroceramides with Transition Metal Ions Bull. Korean Chem. Soc. 2009, Vol. 30, No. 2 399
m/zm/z
m/zm/z
Figure 3. MSn spectra of doubly charged cluster ion, [5DHCer (2)+Mn]2+ (m/z 885) (a) Full-scan MS/MS mass spectrum of [5DHCer(2)+Mn]2+ (b)
Full-scan MS3 (885→714→) mass spectrum of [4DHCer(2)+Mn]2+ (c) Full-scan MS4 (885→714→543→) mass spectrum of [3DHCe r(2)+Mn]2+
(d) Full-scan MS5 (885→714→543→371→) mass spectrum of [2DHCer(2)+Mn]2+.
Scheme 1. Proposed CID (MSn) fragmentation pathway for doubly charged cluster ion, [5DHCer(2)+Mn]2+ (m/z 885.4).
Scheme 2. Proposed CID (MSn) fragmentation pathway for singly charged cluster ion, [3DHCer(6)+Mn+2H2O-H]+ (m/z 1287.0).
0.89 V, 0.96 V, and 1.07 V were required to fragment precursor
ion, [nDHCer(2)+Mn]2+, into several product ions according
to the number of C2-dihydroceramides from five to two,
respectively. This fact indicates that [5DHCer(2)+Mn]2+ was
in the most unstable state than the other doubly charged ions
containing two to four C2-dihydroceramides. The fragmentation
pathway of [5DHCer(2)+ Mn]2+ was proposed in Scheme 1.
The mass spectrum pattern of C2-dihydroceramide with
manganese(II) ion (Fig. 2) is different from that of C2-cera-
mide22 (Fig. 3 in Ref. 22). The peaks of singly charged cluster
ions (Fig. 2) such as [DHCer(2)+Mn+2H2O–H]+, [2DHCer-
(2)+Mn+2H2O–H]+, and [3DHCer(2)+Mn+2H2O–H]+ as well
as doubly charged cluster ions, [nDHCer(2)+ Mn]2+ (n = 2~6)
were distinguishable from those of C2- ceramide.22 Especially,
one of the singly charged ions, [2DHCer(2)+Mn+2H2O–H]+
at m/z 776 was lot more stable than [4DHCer(2)+Mn]2+ at m/z
714 or [5DHCer(2)+Mn]2+ at m/z 865 whereas in the case of
C2-ceramide,22 the most eminent ion [2Cer+Mn+2H2O–H]+
was competitive with [4Cer+ Mn]2+ and [5Cer+Mn]2+. This is
largely because C2-dihydroceramide does not contain carbon
double bond which acts as an acceptor in hydrogen bonds
unlike C2-ceramide. We studied the effect of the structures of
C2-ceramide and C2-dihydroceramide in the complexation
with transition metal ions. The only difference between C2-dihy-
droceramide and C2-ceramide is whether ceramide has double
bond connecting C4 to C5 of the sphingoid backbone or not23

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400 Bull. Korean Chem. Soc. 2009, Vol. 30, No. 2 Jin-Yi Lim et al.
300400500 600 700800
m/z
m/z
900 100011001200 1300
0
20
40
60
80
100
784.1
344.3
748.4
441.3
889.2
1060.6 1126.7
375.2
A : [DHCer(2) + H – H2O] +
B : [DHCer(2) + H] +
C : [2DHCer(2) + Cu] 2+
D : [DHCer(2) + Cu +2H2O - H] +
E : [2DHCer(2) + H] +
F : [2DHCer(2) + Na] +
G : [2DHCer (2)+ Cu - H] +
H : [2DHCer(2) + Cu + 2H2O-H] +
I : [5DHCer(2) + Cu] 2+
J : [6DHCer (2)+ Cu] 2+
K : [3DHCer(2) + Cu + 2H2O - H] +
R
elative A
bundance (%
)
A
326.4
B
C
E
F
G
H
I
687.4
D
709.5
J
K
Relative Abundance (%)
Figure 4. ESI positive ion mass spectrum of aqueous methanol
(25/75%) solution containing a mixture of C2-dihydroceramide
(5.0x10-5 M) dissolved in methanol and copper(II) chloride
(5.0x10-4 M) dissolved in aqueous methanol(50/50%) at 20V tube
lens offset voltage.
and it will be clear that C2-dihydroceramide does not have
carbon double bond connecting through C4 and C5 position
which was present at the same position in C2-ceramide.22
Formation and fragmentation of complexes of C2-dihy-
droceramide with Cu2+. Fig. 4 indicates the full mass spectrum
of C2-dihydroceramide in the existence of copper ion. As
shown in the mass spectrum, the complex ion including
C2-dihydroceramide and copper ion, [2DHCer(2)+Cu+2H2O–
H]+ was dominant. Interestingly, the doubly charged cluster
ions corresponding to the formula of [nDHCer(2)+Cu]2+ were
generated with significantly low abundance. When n is two,
the peak of [2DHCer(2)+Cu]2+ at m/z 375 was too low to be
recognized rather not existed. The fact that the abundance of
[2DHCer(2)+Cu+2H2O–H]+ was remarkable and [nDHCer
(2)+Cu]2+ has extremely low abundance indicates that two
C2-dihydroceramides allow copper ion to be stable by adding
two H2O molecules and losing a proton. Therefore, it can
demonstrate that not [2DHCer(2)+Cu]2+ but [2DHCer(2)+
Cu+2H2O–H]+ was observed with notably great abundance.
The other feature in the complexation of C2-dihydroceramide
with copper ion is that the peaks of [DHCer(2)+H]+ at m/z 344
and [2DHCer(2)+H]+ at m/z 687 have great intensity. It provides
an evidence that enough electron donating groups of C2-dihy-
droceramide assist protonated C2-dihydroceramide itself to be
crucially stable. Furthermore, it tells us that the affinity of
C2-dihydroceramide on copper ion is lower than that of
manganese ion.
C2-dihydroceramide did not yield a wide variety of com-
plexes with copper ion comparing with manganese ion. In
addition, one of the complexed ions produced, [2DHCer (2)+
Cu+2H2O–H]+ was relatively outstanding ion and having the
relative abundance of 100% because copper ion prefers
square-planar when complexing with ligands, not to have
octahedral geometry. The fact that [2DHCer(2)+Cu–H]+ was
also yielded with not low intensity, this might be the other
evidence to prefer copper ion for square-planar. The complex-
ation behavior of C2-dihydroceramide with remaining tran-
sition metal ions, Fe2+, Co2+, Ni2+, and Zn2+ is almost similar to
that of Mn2+, but there is a difference in intensity for complex
ions was observed in all transition metal-C2-dihydroceramide
complexes.
Formation and dissociation of complexes of C6-dihydro-
ceramide with Mn2+. A series of experiments on C6-dihydro-
ceramide with transition metal ions were carried out to explore
the influence of difference in fatty acid on the complexation
with transition metal ions. Overall, the pattern of peaks for
eminent ions generated was similar to that of C2-dihydro-
ceramide. There are various ions of both singly and doubly
charged cluster ions; [DHCer(6)+Mn+2H2O–H]+ at m/z 489,
[2DHCer(6)+Mn+2H2O–H]+ at m/z 888, [3DHCer(6)+Mn+
2H2O–H]+ at m/z 1287, [DHCer(6)+H-H2O]+ at m/z 382,
[DHCer(6)+H]+ at m/z 400, [2DHCer(6)+Na]+ at m/z 821,
[2DHCer(6)+Mn]2+ at m/z 427, [3DHCer(6)+Mn]2+ at m/z
626.5, [4DHCer(6)+Mn]2+ at m/z 826, [5DHCer(6)+ Mn]2+ at
m/z 1025.5, and [6DHCer(6)+Mn]2+ at m/z 1225 (Here, 6 in
parenthesis indicates the carbon number of fatty acid). The
most abundant peak of the spectrum was [2DH Cer(6)+Mn+
2H2O–H]+ at m/z 887, the intensities of singly and doubly
charged species of C6-dihydroceramide were higher than
those of C2-dihydroceramide, and when compared to C2-cera-
mide22 the peak pattern and the intensities of singly and doubly
charged species are different. The ratio of the peak intensity
for [2DHCer(6)+Mn+2H2O–H]+ to that of [nDHCer(6)+Mn]2+
(n = 4 or 5) was reduced as compared with that of C2-dihy-
droceramide, this is due to the influence of steric effect to the
electronic effect of two C6-dihydroceramides was relatively
severe over C2-dihydroceamide. In other words, the electronic
effect of [nDHCer(6)+Mn]2+ (n = 4 or 5) neutralizes the steric
effect of those ions. As the number of C6-dihydroceramides
complexed with manganese ion was above three, we estimate
the surplus C6-dihydroceramides interact with manganese
ion, but not directly rather secondarily through the other
C6-dihydroceamides which have already combined. However,
when the number of C6-dihydroceramide is seven, [7DHCer(6)
+Mn]2+, the peak at m/z 1424 was not shown because the
electronic effect of the seventh C6-dihydroceramide was no
longer able to the influence on manganese ion to be stabilized.
Fragmentation of complexes of C6-dihydroceramide with
Mn2+. In order to confirm the composition of complex ions of
C6-dihydroceramide in presence of manganese ion, and to
monitor the fragmentation pathways of these species, MS/MS
and MSn of those ions have performed. One of the singly
charged species, [3DHCer(6)+Mn+2H2O–H]+ at m/z 1287,
was fragmented into [2DHCer(6)+Mn+2H2O–H]+ at m/z 888
by the loss of one C6-dihydroceramide. MS3 spectrum included
[2DHCer(6)+Mn–H]+ at m/z 852, [2DHCer(6) +Mn–H2O–
H]+ at m/z 834, and [DHCer(6)+Mn+2H2O–H]+ at m/z 489.
Although the MS4 and MS5 were also examined, the spectra
does not illustrate because those are too simple with the lack of
one H2O. The fragmentation pathway of [3DHCer(6) +Mn+2H2O
–H]+ at m/z 1287 was proposed in Scheme 2. MS/MS of
[6DHCer(6)+Mn]2+ at m/z 1225 yields a predominant fragment
ion at m/z 1025.5 corresponding to the formula of [5DHCer(6)
+Mn]2+, by the loss of one C6-dihydroceramide. [4DHCer(6)
+Mn]2+ at m/z 826 was found in MS3 spectrum of [5DHCer(6)
+Mn]2+, of which the precursor ion was [6DHCer(6)+Mn]2+.
MS4 of [4DHCer(6)+Mn]2+ produces the doubly charged ion,

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Complexes of C2- and C6-dihydroceramides with Transition Metal Ions Bull. Korean Chem. Soc. 2009, Vol. 30, No. 2 401
[3DHCer(6)+Mn]2+ at m/z 626.5 by the removal of one C6-dihy-
droceramide. The MS5 spectrum of the precursor ion, [3DHCer(6)
+Mn]2+ was fragmented from [4DHCer(6)+Mn]2+, consists of
singly charged ions such as [DHCer(6)–H2O+H]+ at m/z 382,
[DHCer(6)+H]+ at m/z 400, [2DHCer(6)+Mn–H2O+H]+ at m/z
834, and [2DHCer(6)+Mn+H]+ at m/z 852 together with
[2DHCer(6)+Mn]2+ at m/z 427.
Formation and fragmentation of complexes of C6-dihydro-
ceramide with Cu2+. The full mass spectrum of C6-dihydro-
ceramide (5 ×10-5 M) in the existence of copper ion (5 ×10-4
M) was not so different with C2-dihydroceramide. Surpri-
singly, even though the intensity of [DHCer(6)+H]+ was also
considerably high, the dominant peak in the spectrum was
protonated C6-dihydroceramide, [DHCer(6)+H]+, not one of
the complexed ions. The rationale behind this is that the
synergy between steric effect and electronic effect of C6-
dihydroceramide was produced to make [DHCer(6)+H]+ to be
the most stable form. C6-Dihydroceramide forms a wide variety
of complex ions such as [DHCer(6)+H–H2O]+ at m/z 382,
[DHCer(6)+H]+ at m/z 400, [2DHCer(6)+Cu]2+ at m/z 431,
[DHCer(6)+Cu+2H2O-H]+ at m/z 497, [2DHCer (6)+H]+ at
m/z 799, [2DHCer (6)+Na]+ at m/z 821, [2DHCer (6)+Cu-H]+
at m/z 860, [2DHCer(6)+Cu+2H2O-H]+ at m/z 896, [5DHCer
(6)+Cu]2+ at m/z 1029, [6DHCer(6)+Cu]2+ at m/z 1229, and
[3DHCer(6)+Cu+2H2O-H]+ at m/z 1296. Through the fact that
[2DHCer(6)+Cu-H]+ was found with remarkable abundance
as well as [2DHCer(6)+Cu+2H2O-H]+, the preference of
copper ion for square-planar is confirmed. Moreover, the ratio
of metal complex ions to C6-dihydroceramide adduct not
including copper ion is lower than C2-dihydroceramide, due
to the steric effect of fatty acid from C6-dihydroceramide.
Since the affinity of C6-dihydroceramide for copper ion is not
stronger than manganese ion, some peaks relating copper ion
were seen with rather trivial intensity like [2DHCer(6)+Cu]2+
(When the tube lens offset voltage of 10 V was applied and the
abundance of this ion was nearly below 5 %). The complexation
of C6-dihydroceramide with other metal ions like Fe2+, Co2+,
Ni2+ and Zn2+ is not mentioned since the complexation with
those ions was not much different from that of Mn2+.
Conclusions
In the present work, the complexes and their fragmentation
pathways of C2- and C6-dihydroceramides with transition
metal cations (Mn2+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2+) were
explored by ESI-MS/MS. All of the above mentioned metal
ions complex with C2- and C6-dihydroceramides to form
doubly charged cluster ions as well as singly charged cluster
ions containing metal ion and C2- or C6-dihydroceramides.
Although extreme distinction in the full mass spectrum
between C2-dihydroceramide and C6-dihydroceramide in
existence of transition metal is not seen, the most high
abundant peaks of C6-dihydroceramide, [DHCer(6)+H]+ and
[2DHCer (6)+H]+, which are not complexed to copper, were
observed. While the most dominant peak of C2-dihydro-
ceramide, [2DHCer(2)+Cu+2H2O–H]+, was the complexed
ion including copper. The reason for the difference in
complexation with copper between C2-dihydroceramide and
C6-dihydroceramide is that the steric effect due to the
difference in carbon number of fatty acid in dihydrocer-
amides. That is, the steric effect of C6-dihydroceramide
outweighs the electronic effect of C6-dihydroceramide. For
copper(II), the most common coordination numbers are 4, 5
and 6, but tetragonal distorted octahedral geometries is not by
far different from square-planar while the other metal ions
prefer the octahedral geometry.24 In sum, the unique mass
spectrum of copper(II) in the complexation with ceramide
results from low ceramide’s affinity on Cu(II) and distinctive
coordination geometry. Through the experiments performed
in this paper, the fact that the tandem mass spectrometry is a
rapid, sensitive, and suitable method to investigate the
complexation of several ceramides with transition metal ions
is proved.
Acknowledgments. The authors gratefully acknowledge
the support by the Korea Research Foundation (Grant No.
KRF-2007-J00903) and Changwon National University (2007).
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