RAPID COMMUNICATIONS IN MASS SPECTROMETRY
Rapid Commun. Mass Spectrom. 2006; 20: 137–150
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.2266
Analysis of urinary nucleosides. V. Identification
of urinary pyrimidine nucleosides by liquid
chromatography/electrospray mass spectrometry
Alison Bond1,5, Edward Dudley1, Filip Lemiere2,3, Robin Tuytten1,2, Salah El-Sharkawi4,
A. Gareth Brenton1,6, Eddy L. Esmans2,3 and Russell P. Newton1,5*1Biomolecular Analysis Mass Spectrometry (BAMS) Facility, Grove Building, University of Wales Swansea, Singleton Park,
Swansea SA2 8PP, UK2Department of Chemistry, Nucleoside and Mass Spectrometry Research Unit, University of Antwerp, Groenenborgerlaan 171, B-2020,
Antwerp, Belgium3Centre for Proteome Analysis and Mass Spectrometry (CeProMa), University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium4Oncology Unit, Singleton Hospital Trust, Sketty, Swansea SA2 8QA, UK5Biochemistry Group, School of Biological Sciences, Wallace Building, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK6Mass Spectrometry Research Unit, Department of Chemistry, Grove Building, University of Wales Swansea, Singleton Park,
Swansea SA2 8PP, UK
Received 4 October 2005; Revised 24 October 2005; Accepted 24 October 2005
Modified urinary nucleosides are potentially invaluable in cancer diagnosis, as they reflect altered
RNA turnovers. High-performance liquid chromatography (HPLC) was combined with full-scan
mass spectrometry, tandem mass spectrometry, MSn analysis and accurate mass measurements in
order to identify pyrimidine nucleosides purified from urine. Potential nucleosides were assessed
by their evident UV absorbance in the HPLC chromatogram and then further examined by the
various mass spectrometric techniques. In this manner numerous pyrimidine nucleosides were
identified in the urine samples from cancer patients including pseudouridine, cytidine, two
methylcytidines and an acetylcytidine. Furthermore, a number of novel modified pyrimidine
nucleosides were tentatively identified via critical interpretation of the combined mass spectro-
metric data. Copyright # 2005 John Wiley & Sons, Ltd.
A number of past studies have indicated that modified
nucleosides excreted in the urine have the potential to act as
cancer biomarkers with increased levels arising from disease
onset and progression.1–4 Modified nucleosides differ from
‘normal’ nucleosides usually by a minor modification, with
the most common alteration being methylation. Modification
of nucleosides occurs after incorporation into nucleic acid
molecules of which transfer RNA (tRNA) is the most com-
monly modified. During tRNA turnover these compounds
are released from the nucleic acid molecules and, due to the
absence of salvage or degradation pathways and the potential
toxicity of their accumulation, they are excreted via the urine.
Although the levels of these compounds have been studied
over a number of decades it is only recently that mass spectro-
metric means have been employed for the purpose.5–10
Mass spectrometry (MS) has been successfully applied to
analyse nucleosides from various sources including ancient
seeds11 and RNA hydrolysates12 and has been assessed by
our group for the detection of the urinary modified nucleo-
sides. Electrospray ionisation tandem mass spectrometry
(ESI-MS/MS), when coupled with high-performance liquid
chromatography (HPLC), has the advantage over previously
utilised methods of detection, which include HPLC with UV
absorbance detection,1,2 capillary electrophoresis13 and
fluorescence spectroscopy,14 that more structural data can
be obtained, thereby allowing improved certainty on the
identity of the nucleosides being studied. The need to employ
MS analysis as opposed to HPLC alone can readily be gauged
once the number and diversity of naturally occurring
modified nucleosides are appreciated, as detailed in two
previous reviews.15,16 Nucleoside identification by UV
absorbance and HPLC retention time, typically over an hour
run time, can clearly lead to co-elution and erroneous
identification if such a large number of similar compounds
is present. MS analysis will allow the discriminative detection
of co-eluting nucleosides as long as they differ (sufficiently)
in mass. Therefore, using MS it is less likely that the results
from two co-eluting nucleosides will accidentally be assigned
as being that of one nucleoside. Furthermore, many mass
spectrometers can acquire product ion spectra of the nucleo-
sides and these can provide structural information on these
compounds. The fragmentation of these compounds and
their protonated heterocyclic base units [BHþ] by MS means
Copyright # 2005 John Wiley & Sons, Ltd.
*Correspondence to: R. P. Newton, Biochemistry Group, School ofBiological Sciences, Wallace Building, University of WalesSwansea, Singleton Park, Swansea SA2 8PP, UK.E-mail: [email protected]/grant sponsor: Evan Davies Trust Fund.Contract/grant sponsor: Flemish Government GOA Action;contract/grant numbers: FWO-Vlaanderen 1.5.139.00 andG.2133.94.
has been reported in the past,17 and additional insights into
the fragmentation pathways were acquired using specific
isotopically labelled nucleosides.18,19 These pathways allow
the assignment of characteristic fragments that arise from
specific modifications and this therefore makes MS an
efficient tool in the identification of modified nucleosides.
The use of an ion trap mass spectrometer in this study allows
the further controlled fragmentation of the product ions
produced by the initial MS/MS experiment in subsequent
MSn experiments as long as the signal of the product ions is
sufficient. Previous application of this methodology has shown
the presence of 50-deoxycytidine in a head and neck cancer
patient,9 the first demonstration of the occurrence of this
modified nucleoside in a mammalian species, and of the
modified purines, and adenosine, 1-methyladenosine, xantho-
sine, N1-methylguanosine, N2-methylguanosine, N2,N2-
dimethylguanosine, N2,N2,N7-trimethylguanosine, inosine,
and 1-methylinosine in urine samples from cancer patients.22
The aim of the present study was to utilise HPLC/ESI-MS to
study modified pyrimidine nucleosides purified from a
number of urine samples pooled together and to examine the
fragmentation of these compounds under MS/MS and, if
possible, MSn conditions in order to determine the identity of
the nucleosides present. Where appropriate accurate mass MS,
in which the mass of the ion is determined to such a degree that
the empirical formula of the ion studied can be determined,
was also utilised. Constant neutral loss (CNL) scanning was
also used to aid in determining the nature of the sugar residue
and whether it was modified.
EXPERIMENTAL
MaterialsAll the chemicals used were purchased from the Sigma-
Aldrich Company Ltd. (Poole, UK) except HPLC-grade
methanol and water from Fisher Scientific (Loughborough,
UK). Water for the HPLC purification experiments was pre-
pared in-house using an Elix-water purification system
(Millipore, Watford, UK).
Urine samplesUrine samples were obtained from the Oncology Unit of the
Singleton Hospital, Swansea, UK. Typically, 10–20 mL sam-
ples were obtained of which 5 mL were used in the purifica-
tion process. Samples not processed upon collection were
frozen and stored at �208C until use.
Urinary nucleoside purificationThe pyrimidine nucleosides were purified from the urine
(after addition of an internal standard, tubercidin) using a
method previously developed and reported by our group20
utilising a cis-diol affinity column followed by a combination
of two ion-exchange columns. The purification resulted in
two fractions containing purified pyrimidine nucleosides.
One fraction contained the more basic nucleosides, such as
cytidine and adenosine, and the second fraction contained
the less basic nucleosides such as uridine and its derivatives.
HPLC/ESI-MS analysisThe separation of the pyrimidine nucleosides by HPLC and
the ionisation of these compounds were optimised as
described previously.21,24 A Hewlett Packard 1100 HPLC sys-
tem (Hewlett Packard, Amstelveen, The Netherlands) was
used with a 150� 4.6 mm i.d. Spherisorb C18 HPLC column
(Jones Chromatography, Hengoed, UK) for the separation of
the nucleosides. The mobile phases used were (a) 5 mM
ammonium acetate (pH 5.0) and (b) methanol at a flow rate
of 0.2 mL/min. The gradient was held at 5% B for 5 min before
being increased linearly to 10% B by 10 min, to 35% B by
30 min, held at 35% B for 10 min and then the column was
re-equilibrated in 5% B for 15 min. The eluent was passed
via a UV cell, monitoring at 254 nm, to an LCQ ion trap
mass spectrometer with an ESI source (ThermoElectron,
Hemel Hempstead, UK). Between the UV cell and the mass
spectrometer a divert valve was employed in order to divert
to waste the salts eluting before the nucleosides. The ESI
source was operated in positive ion mode with optimised
conditions as previously reported.21 The mass spectrometer
recorded full-scan mass spectra over the mass range
m/z 50–800 accumulating three microscans, using a maxi-
mum injection time of 200 ms and an electron multiplier
voltage of 1200 V. The pyrimidine nucleosides were
identified based on their UV absorbance, the presence of their
protonated molecule, together with ions arising from their
partial in-source fragmentation and the addition of a
sodium cation.
HPLC/ESI-MS/MS and -MSn
The HPLC and ESI conditions were as described above.
During MS/MS experiments the protonated molecule was
chosen as the precursor ion and isolated in the ion trap.
Next, a collision energy (represented as a percentage of a
maximum possible energy) sufficient to fragment the
majority, if not all, of the precursor ions was used to pro-
duce product ion spectra of the pyrimidine nucleosides.
In MSn experiments the process was further repeated using
the most abundant product ion as the new precursor ion.
HPLC purification and accurate mass MSPrior to accurate mass determination the pyrimidine nucleo-
sides were separated on an 150� 4.6 mm i.d. APEX II HPLC
column (Jones Chromatography) at a flow rate of 0.5 mL/min
by an 1100 HPLC system (Hewlett Packard). The mobile phase
consisted of an aqueous mobile phase A (5 mM ammonium
acetate, pH 5.0) and an organic mobile phase B (methanol).
The gradient applied was to run from 5% B to 10% B from
0 to 7.5 min and then from 10% B to 30% B from 7.5 to
23.5 min. Fractions were collected, examined by accurate MS,
and correlated to the original HPLC/MS data by the corre-
sponding m/z values detected. Accurate mass determination
was carried out on a Q-ToF Ultima mass spectrometer (Waters,
Manchester, UK) using the nanolockspray source.The fractions
were infused at 1mL/min through the analyte sprayer while a
reference compound was sprayed using the reference sprayer,
also at a flow rate of 1mL/min. When necessary the concentra-
tions of the analyte and the reference compound were adjusted
in order to reduce their signal to less than 200 counts/s scan
time and thereby avoid any deadtiming effect that would
reduce mass accuracy.
The reference compounds varied but were usually chosen
to produce a reference ion close in mass/charge ratio to the
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 137–150
138 A. Bond et al.
expected analyte ion; generally unmodified nucleosides and
their bases known not to occur in the analyte were used. The
Q-ToF was operated in positive ion mode with a spray
voltage of 3 kV, a source temperature of 808C, a cone gas flow
of 50 L/h, a cone voltage of 100 V, a nanoflow gas setting of
10 psi, and the MCP voltage set to 2100 V. For full-scan
spectra the collision energy was set to 10 eV and for MS/MS
experiments it was first adjusted to obtain the best data
manually prior to automatic collection of the MS/MS data
with lock mass.
Constant neutral loss (CNL) experimentsCNL spectra were obtained on a Quattro II triple-quadrupole
MS system (Waters) with capillary voltage at 3.64 kV, cone
voltage 20 V, and source temperature of 808C, as previously
described.24 CNL spectra of 132 and 116 Da (loss of ribose
and deoxyribose, respectively) were monitored by scanning
the first mass analyser of the triple-quadrupole MS system
over a mass range of m/z 132–450 and the second mass ana-
lyser over the appropriate mass range—m/z 116–334 for a
CNL of 116 Da. The scan time was 1.49 s. The collision gas
pressure was 3.5� 10�3 mbar argon, and the collision energy
was 20.0 eV.
RESULTS AND DISCUSSION
Figure 1 shows UV traces (monitored at 254 nm) of the puri-
fied nucleosides from the basic nucleosides fraction (Fig. 1(a))
and the less basic nucleosides fraction (Fig. 1(b)) separated by
the HPLC conditions applied. The peaks numbered represent
those that are suspected as arising from pyrimidine nucleo-
sides. Many of the putative modified pyrimidine nucleosides
were confirmed by the fragmentation patterns arising from
their MS/MS and MSn analysis, accurate mass data and com-
parison with the response of purchased pure compounds to
these experiments. For a number of nucleosides no commer-
cial standard was available and so only the MS analysis could
be used in order to determine the identification of these
nucleosides. The nucleosides for which a commercial sample
was obtained will be discussed first, detailing the analysis of a
Figure 1. UV trace of HPLC indicating pyrimidine nucleosides in (a) the more basic nucleoside fraction
(b) the less basic nucleoside fraction.
MS analysis of urinary nucleosides. V: pyrimidine nucleosides 139
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 137–150
m/z100 200 300 400 500 600 700
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
R
elat
ive
Abu
ndan
ce
Rel
ativ
e A
bund
ance
245.0
80 100 120 140 160 180 200 220 240 260 280 3000
10
20
30
40
50
60
70
80
90
100209.0
178.8
227.3245.2
m/z
80 100 120 140 160 180 200 220 240 260 280 3000
10
20
30
40
50
60
70
80
90
100209.0
178.8
227.3245.2
m/z
(a)
(b)
(c)
60 80 100 120 140 160 180 200 220 240 0
10 20 30 40 50 60 70 80 90
100 191.1
125.0
209.1
m/z
Figure 2. MS analysis of peak 10: (a) full-scan ion trap mass spectrum; (b) MS/MS product
ion spectrum of m/z 245; and (c) MS3 spectrum of m/z 245! 209.
140 A. Bond et al.
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 137–150
single pyrimidine nucleoside in detail and summarising the
data for the remaining compounds.
Identification of urinary modified pyrimidinenucleosides with available commercial standards
HPLC-UV peak 10Peak 10 (Fig. 1(b)) occurs in the less basic nucleoside fraction
and elutes at 13.0 min. The background-subtracted spectrum
consists of a base peak atm/z 245 which has a co-eluting ion at
m/z 209 (Fig. 2(a)). MS/MS of the ion at m/z 245 (as shown in
Fig. 2(b)) produces product ions at m/z 209 (100% relative
abundance), 227 (8%) and 179 (15% relative abundance).
These product ions represent loss of a single water molecule
from the precursor ion (to yield m/z 227), loss of two water
molecules (to m/z 209) and loss of two water molecules and
CH2O (to m/z 179), as shown in Fig. 3. Further fragmentation
by MS3 analysis of the product ion at m/z 209 (Fig. 2(c)) gave
rise to major ions at m/z 191 (a further loss of water) and 125
(thought to follow from further fragmentation of the ribose
ring with a single carbon remaining attached to the base).
The accurate mass data (m/z 245.0770) confirmed the empiri-
cal formula of the protonated nucleoside, corresponding to
pseudouridine or uridine (C9H13N2O6, calc. m/z 245.0774).
The product ion spectra of the nucleoside, however, distin-
guished the nucleoside as pseudouridine and reconfirmed
the nature of the product ions at m/z 209 and 191 as detailed
in a recent publication by our group27 (see Table 1). The
obtained data was identical to that obtained from the com-
mercially available pseudouridine. Therefore peak 10 is con-
firmed as this nucleoside (as indicated in Fig. 3).
Pseudouridine is known to be a relatively common modified
nucleoside and it has been isolated from eukaryotic extracts
in the past.23 The fragmentation of pseudouridine differs
from that usually exhibited by other nucleosides. During
MS/MS analysis the bond between the nucleobase and ribose
sugar is not broken and hence the primary product ion is not
the protonated base, as is common in the other nucleosides.
This difference is attributed to the availability of a more
stable C–C bond between the sugar and base rather than
the more common C–N bond. Instead, consecutive losses of
water are seen (breakdown of the ribose moiety) followed by
the suspected loss of a CH2O section and further fragmenta-
tion of the sugar (Fig. 3).27
HN NH
O
O
H+
O
OHHO
HOH2C
-H2O
-2 H2O
- 2H2O + CH2O
HN NH
O
O
H+
O
OHH
H2C
-H2O
m/z 209
m/z 191
HN NH
O
O
+CH2
m/z 125
m/z 179
m/z 245
m/z 227
Figure 3. Fragmentation of peak 10, protonated pseudour-
idine.
Table 1. Identification of pyrimidine nucleosides for which commercial standards were available: (a) ion trap MS/MS and MSn
data and (b) Q-ToF accurate mass analysis data
UV Peak Identification Ion seen (Th) [identity] MS/MS (Th) [identity] MSn (Th) [identity]
(a)1 Cytidine 244 [MHþ] 112 [BHþ] 95 [BHþ–NH3]4 5-Methylcytidine 258 [MHþ] 126 [BHþ], 240 [MHþ–H2O] n/a9 N-Acetycytidine 286 [MHþ] 154 [BHþ], 112 [cytosineHþ] 112 [cytosineHþ] 95
[cytosineHþ–NH3]10 Pseudouridine 245 [MHþ] 227 [MHþ–H2O] ,
209 [MHþ–2H2O],179 [MHþ–2H2OþCH2O]
191 [MHþ–3H2O],125 [BHþþCH2]
(b)UV Peak Identification Empirical formula of ion seen
(scan type) [identity]Theoretical
(m/z)Experimental
(m/z)Mass difference
(ppm)
1 Cytidine C9H14N3O5 (Full scan) [MHþ] 244.0933 244.0940 2.7C4H6N3O (MS/MS) [BHþ] 112.0511 112.0515 3.7
4 5-Methylcytidine C10H16N3O5 (Full scan) [MHþ] 258.1090 258.1103 5.1C5H8N3O (MS/MS) [BHþ] 126.0667 126.0660 �5.9
9 N-Acetycytidine C11H16N3O6 (Full scan) [MHþ] 286.1039 286.1035 �1.5C6H8N3O2 (MS/MS) [BHþ] 154.0617 154.0640 15.0
10 Pseudouridine C9H13N2O6 (Full scan) [MHþ] 245.0774 245.0770 �1.5C9H9N2O4 (MS/MS)[MHþ–2H2O],
209.0562 209.0554 �3.8
C9H7N2O3 (MS/MS)[MHþ–3H2O],
191.0457 191.0453 �1.9
C8H7N2O3 (MS/MS)[MHþ–2H2OþCH2O]
179.0457 179.0466 5.0
C5H5N2O2 (MS/MS)[BHþþCH2]
125.0351 125.0361 8.1
MS analysis of urinary nucleosides. V: pyrimidine nucleosides 141
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 137–150
The other pyrimidine nucleosides for which standards
were available for comparison, along with the data obtained,
are summarised in Table 1. These nucleosides are cytidine,
5-methylcytidine and N4-acetylcytidine. It should be noted
that UV peak 9 was shown to contain two nucleoside-related
ions, only one of which is shown in the table (the second ion
present had no comparable standard available and so is
discussed later in this paper). The MS data allows the
determination of the identities of all the nucleosides listed in
Table 1. All except pseudouridine exhibit the most common
product ions formed by MS/MS analysis of nucleosides (the
loss of 132 Da due to the cleavage of the glycosidic bond). All
the data shown in Table 1 were reproduced with the standard
compounds and at the same time the retention behaviour on
the HPLC system used was shown to be identical between the
suspected nucleosides and standards. The accurate mass data
for some of the product ions is markedly less accurate than
that for the precursor ion, most probably due to the low levels
of the compounds available in the urine. Therefore, ion
statistics were not ideal to produce high-quality data. Despite
this, the top listed calculated elemental compositions were
very sensible, indicating the limitations of expressing mass
accuracy in ppm at low m/z ratios.
Identification of urinary modified pyrimidinenucleosides with no available commercialstandardsThe identification of the nucleosides for which no comparable
standards were available relied on the MS data alone to elu-
cidate the suspected structures of the UV peaks detected. The
reliability of these identifications and the use of the MS data
alone to elucidate the suspected structures of the nucleosides
detected by UV are discussed later.
Peak 2Peak 2 eluted at 17.0 min and the background-subtracted
spectrum shows co-eluting ions at m/z 228 and 112 with rela-
tive abundances of 100 and 10%. Fragmentation of m/z 228
yields product ions at m/z 210 and 112 with relative abun-
dances of 10 and 100% (data not shown). The data suggest
that the compound is a deoxycytidine. The m/z 228 ion is
therefore from the protonated molecule with the m/z 112
ion being formed by the fragmentation of m/z 228 to give
the protonated cytosine base. Constant neutral loss of
132 Da gave no peak, but CNL of 116 Da gave a peak at
16.8 min arising from a precursor ion of m/z 228 (Fig. 4(a)).
This contrasts with the spectra for cytidine with no CNL
peak for loss of 116 Da but a peak at 11.8 min representing a
CNL of 132 Da from a precursor ion at m/z 244 (Fig. 4(b)),
confirming that peak 2 contains a deoxyribose moiety.
Fragmentation yields the protonated base also and a loss of
water (tom/z 210), as was also seen in the commercially avail-
able 20-deoxycytidine. The peak co-elutes with the standard
20-deoxycytidine; however, such a compound would not be
expected to be retained upon the Affi-Gel 601 affinity column
used during purification of the nucleosides as this column
would only bind nucleosides with a cis-diol group. The
work described here, along with additional experiments, as
detailed in a previous publication, identified the nucleoside
as 50-deoxycytidine.9 The newly available accurate mass
data for the nucleoside and the empirical formulae obtained
experimentally for the intact nucleoside (m/z 228.0973) and its
product ions (m/z 112.0500 and 210.0895), the cytosine base
and loss of water from the ion, were shown to match the the-
oretical formulae for a protonated deoxycytidine
(C9H14N3O4, m/z 228.0984), protonated cytosine (C4H6N3O,
m/z 112.0511) and the protonated deoxycytidine minus water
(C9H12N3O3, m/z 210.0879), see Table 2(b).
Peak 5The peak at 23.2 min gives a spectrum in which ions atm/z 258
and 112 co-elute, with m/z 258 predominating in the full-scan
mass spectrum. The collision-induced dissociation (CID) of
the ion atm/z 258 produces an ion atm/z 112 (data not shown).
The ion at m/z 258 matches that of a protonated methylcyti-
dine and the m/z 112 ion matches that of an unmodified, pro-
tonated cytosine base. The CNL chromatogram for loss of
132 Da between m/z 257.5 and 258.5 shows a peak at
18.83 min (Fig. 5) corresponding to peak 4 in Fig. 1(a) (as
described previously in this paper), but no peak at 23.2 min
(peak 5), nor any peaks for CNL 116, indicating that peak 5
contains a modified ribose, suggesting that the position of
methylation is on the sugar not on the base. Fragmentation
of the methylcytidine ion at m/z 258 gave an unmodified
base adding further weight to the proposition that the cyti-
dine nucleoside must be methylated on the sugar moiety.
The accurate mass analysis (Table 2(b)) confirms that the for-
mulae of both the m/z 258 and the 112 ions are as described
above. The only isomer detected in eukaryotes in the past is
20-O-methylcytidine.25 This nucleoside again would not be
expected to be retained upon an Affi-Gel 601 column and
when the standard compound was tested this was shown to
be true. It is therefore concluded that the nucleoside is 50-O-
methylcytidine, which as it is methylated on the sugar moiety
still retains the cis-diol group. Further product ion spectra
were not attainable due to insufficient signal. Also, as CID
Figure 4. Constant neutral spectra of peak 2: (a) CNL of
116Da, scanned between m/z 227.5 and 228.5 and (b) CNL
of 132Da, scanned between m/z 243.5 and 244.5.
142 A. Bond et al.
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 137–150
of the m/z 258 ion produced the protonated cytosine base as
the sole product ion with the suspected protonated methyl-
ribose sugar absent from the product ion spectrum (due to
the more favourable proton affinity of the nucleobase than
of the sugar), it is expected that further fragmentation analy-
sis would offer no further information for the identity of the
nucleoside.
Peak 7Peak 7 elutes at 27.8 min and the spectrum shows co-eluting
ions at m/z 272 and 112 (Fig. 6(a)). The MS/MS fragmentation
of the m/z 272 ion is shown in Fig. 6(b) and indicates only one
product ion at m/z 112, identical to that of a protonated cyto-
sine base. Three possibilities were considered for the com-
pound based on this data. The mass of the protonated
molecule suggested dimethylated cytidine; however, this
would seem unlikely given the MS/MS results as they sug-
gest, if this identification were correct, that either both the
methyl groups are cleaved from the base during fragmenta-
tion or that both the methyl groups are situated on the sugar.
It was considered unlikely that both methyl groups would be
lost at once and also unlikely that the methyl groups are both
on the sugar. The latter would require methylation at one of
the carbons essential for the formation of a cis-diol group
used for affinity purification of the nucleosides. This sugges-
tion was therefore discounted as a compound modified in
such a way would not be purified during the procedure
used for the extraction of the nucleosides from the urine.
The second and third possible structures were ethylcytidine
and formylcytidine. The MS/MS data suggested either that
the ribose and the extra group (be it a formyl or an ethyl moi-
ety) would be simultaneously lost upon fragmentation or
alternatively that the extra group must be situated on the
sugar and lost when the sugar base bond is cleaved. The first
idea seemed unlikely as fragmentation of the standard
nucleosides cleaved only the glycosidic bond producing the
protonated base (with the exception of pseudouridine). The
CNL chromatogram(132 Da) (Fig. 6(c)) shows no peak at
Table 2. Identification of pyrimidine nucleosides for which no commercial standards were available: (a) ion trap MS/MS and MSn
data and (b) Q-ToF accurate mass analysis data
(a)
UV Peak Identification Ion seen (Th) [identity]MS/MS (Th)
[identity] MS3 (Th) [identity]MS4 (precursorion) [identity]
2 50-Deoxycytidine 228 [MHþ] 112 [BHþ], 210 n/a n/a5 50-O-Methylcytidine 258 [MHþ] 112 [BHþ] n/a n/a7 50 Formylcytidine 272 [MHþ] 112 [BHþ] n/a n/a3 Methylacetylcytidine 300 [MHþ] 168 [BHþ], 112
[cytosineHþ]150 [BHþ-H2O], 112[cytosineHþ], 95[cytosineHþ-NH3] (168)
95 [cytosineHþ–NH3] (150)
6 Ethylacetylcytidine 314 [MHþ] 182 [BHþ], 112[cytosineHþ]
164 [BHþ–H2O], 112[cytosineHþ], 95[cytosineHþ–NH3] (184)
95 [cytosineHþ–NH3] (164)
8 PCNR 271 [MHþ] 139 [BHþ] 121 [BHþ–H2O] (139) n/a9 PCNR 271 [MHþ] 139 [BHþ] 121 [BHþ–H2O] (139) n/a
(b)
UV Peak Identification Empirical formula of ionseen (scan type) [identity]
Theoretical(m/z)
Experimental(m/z)
Mass difference(ppm)
2 50-Deoxycytidine C9H14N3O4 (Full scan) [MHþ] 228.0984 228.0973 �5.0C4H6N3O (MS/MS) [BHþ] 112.0511 112.0500 �9.7C9H12N3O3 (MS/MS) 210.0879 210.0895 7.8
5 50-O-Methyldeoxycytidine C10H16N3O5 (Full scan) [MHþ] 258.1090 258.1079 �4.3C4H6N3O (MS/MS) [BHþ] 112.0511 112.0510 �0.9
7 50-Formylcytidine C10H14N3O6 (Full scan) [MHþ] 272.0883 272.0901 6.8C4H6N3O (MS/MS) [BHþ] 112.0511 112.0513 2.3
3 Methylacetylcytidine C12H18N3O6 (Full scan) [MHþ] 300.1196 300.1185 �3.7C7H10N3O2 (MS/MS) [BHþ] 168.0773 168.0784 6.7
6 Ethylacetylcytidine C13H20N3O6 (Full scan) [MHþ] 314.1352 314.1323 �9.2C8H12N3O2 (MS/MS) [BHþ] 182.0930 182.0929 �0.3
8 PCNR C11H15N2O6 (Full scan) [MHþ] 271.0930 271.0957 8.0C6H7N2O2 (MS/MS) [BHþ] 139.0508 139.0494 �9.7C6H4NO2 (MS/MS) [BHþ–NH3] 122.0242 122.0245 2.1
9 PCNR C11H15N2O6 (Full scan) [MHþ] 271.0930 271.0956 9.7
Figure 5. Constant neutral loss spectrum of peak 5: CNL of
132Da, scanned between m/z 257.5 and 258.5.
MS analysis of urinary nucleosides. V: pyrimidine nucleosides 143
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 137–150
Figure 6. MS analysis of peak 7: (a) full-scan ion trap mass spectrum; (b) MS/MS of m/z 272; and (c) CNL
of 132Da, scanned between m/z 271.5 and 272.5.
144 A. Bond et al.
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 137–150
27.8 min indicating that the modification is on the ribose
group: this extra group must again be present at the 50-posi-
tion as the compound was retained upon the Affi-Gel 601 col-
umn. Accurate mass data obtained both from the full-scan
spectrum and from the product ion scans of m/z 272 showed
that the nucleoside was a formylcytidine as the empirical for-
mulae of m/z 272 and 112 matched those of a formylcytidine
and a cytosine base (see Table 2(b)). Therefore, the other
options, dimethylcytidine and ethylcytidine, could be dis-
counted. The peak is therefore considered to be 50-formylcy-
tidine and the structure of this nucleoside and its proposed
fragmentation are given in Fig. 7.
Peak 3Peak 3 has a retention time of 18.0 min and the spectrum indi-
cates a sole ion at m/z 300 (Fig. 8(a)). The fragmentation of
m/z 300 and further product ion spectra are shown in
Figs. 8(b), 8(d) and 8(e). The MS/MS spectrum of m/z 300
(Fig. 8(b)) shows the simple loss of 132 Da to give a product
ion at m/z 168 and this is concluded to be due to a loss of a
ribose sugar as seen in other nucleosides. The presence of
an unmodified ribose sugar is confirmed by the CNL chroma-
togram (of 132 Da) showing a peak at 18.0 min (Fig. 8(c));
interestingly a second peak is evident at 26.8 min, corre-
sponding to HPLC peak 6, as will be discussed later. Frag-
mentation of m/z 168 produces an ion representing a loss of
water at m/z 150 and ions at m/z 112 and 95 (Fig. 8(d)). These
latter two ions are commonly seen in the fragmentation of
cytidine and its derivatives and represent the protonated
cytosine base and its deamination product. Further fragmen-
tation of the ion at m/z 150 (Fig. 8(e)) yields a product ion at
m/z 95 representing a fragmentation of a possible cytosine
base. Peak 3 is therefore considered to be a cytidine deriva-
tive. As fragmentation of m/z 168 gives the unmodified cyto-
sine base (m/z 112) it is thought that the modification of the
cytidine must be a single group rather than a number of smal-
ler modifications such as methylations. Possible nucleosides
with an appropriate m/z value are a dimethylated formylcy-
tidine or a singly methylated acetylcytidine. The latter is
thought to be more likely as the loss of two methyl groups
as well as a formyl group during MS3 is less likely than loss
of a methyl group and acetyl group which may both be pre-
sent on the nitrogen at position 4 of the cytosine base. Accu-
rate mass data for the precursor ion (m/z 300.1185) and the
main product ion in the MS/MS spectrum (m/z 168.0784),
as shown in Table 2(b), again confirmed that the nucleoside
is a methylated acetylcytidine (theoretical m/z 300.1196 and
168.0773) rather than a dimethylated formylcytidine and
hence add further evidence for the structures shown in
Fig. 9, which indicates the suspected fragmentation pathway
of the putative methylated acetylcytidine.
Peak 6Peak 6 elutes at 26.8 min and its spectrum shows an ion atm/z
314 co-eluting with the internal standard tubercidin (m/z 267;
Fig. 10(a)). MS/MS, MS3 and MS4 fragmentation data for the
ion atm/z 314 are summarised in Fig. 11. The spectra of them/z
314 ion shows first an ion atm/z 182 corresponding to a loss of
a ribose sugar to give a protonated base moiety (assuming
that the compound is a nucleoside). The reconstructed ion
chromatogram for m/z 314 of the CNL spectrum (loss of
132 Da, Fig. 10(b)) shows a single peak at 26.8 min corre-
sponding to this peak 6.
Interestingly, the reconstructed ion chromatogram for m/z
286 of the same CNL analysis (Fig. 10(c)) shows three peaks at
18.1, 26.7 and 34.1, respectively, corresponding to HPLC
peaks 3, 6 and 9, suggesting that peak 6 ([MHþ] at m/z 314)
and peak 3 ([MHþ] atm/z 300) can both yield an ion atm/z 286
capable of then losing the ribose sugar. As noted above, a
structural similarity between HPLC peaks 3 and 6 was
already indicated by the presence of two peaks in the CNL
spectrum (132 Da) from HPLC peak 3 ([MHþ] at m/z 300) at
m/z 299.5–300.5, at retention times identical to those of peaks
3 and 6 (Fig. 8(c)). Peak 9 has already been identified as
acetylcytidine (Table 1). It seems that peak 6 (m/z 314) can give
rise to ions at m/z 300 and 286 and peak 3 (m/z 300) can give
rise to an ion at m/z 286. These ions are formed before the first
quadrupole in the triple-quadrupole instrument and
undergo a loss of 132 (ribose) during MS/MS analysis. The
loss of 14 Da from these compounds is difficult to explain; one
current possibility is that unusual losses and adduct
formations occur in the gas phase, as previously observed
in the nucleoside guanosine by our group,28 although the
exact nature of these is not easily determined from
the available data. An alternative explanation would be the
chemical alteration of the nucleoside in solution in the dead
volume space between the HPLC column and the first
quadrupole, possibly the replacement of the methyl or ethyl
group with a hydrogen atom donated from the mobile phase.
Due to the small quantities of sample available because of
their clinical nature, insufficient sample remained in order to
test these explanations; however, it is felt that as a possible
characteristic of these types of modified nucleoside they may
offer value as ions that indicate the presence of this type of
related modification.
In the MS/MS spectrum of peak 6 a second product ion at
m/z 112 is detected with a relative abundance of 10%. Further
fragmentation of the m/z 182 ion from this compound (loss of
ribose) results in the loss of water to give an ion atm/z 164 and
ions at m/z 112 and 95, which are commonly seen in the
fragmentation spectra of cytidine and its derivatives and
were identical in nature to those seen in the fragmentation of
peak 3. Fragmentation of the ion atm/z 164 gives a product ion
at m/z 95 (again possibly relating to cytosine minus an
NH2
O
N
NO
OH OH
C-OH2C
N
NO
NH2
m/z 112
m/z 272
H
O
m/z 272
Figure 7. Fragmentation of peak 7, protonated 50-formylcy-
tidine.
MS analysis of urinary nucleosides. V: pyrimidine nucleosides 145
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 137–150
ammonia group). This data suggests that the compound is a
derivative of cytidine. It is also considered to be a further
methylated isomer of peak 3 as the fragmentation losses
mimic this nucleoside and the difference in molecular weight
of the two compounds is only 14 Da, corresponding to the
replacement of a hydrogen atom with a methyl group. If this
assumption is correct such a methylation would probably
occur on the methyl group already present and so the
nucleoside would be provisionally identified as ethylacetyl-
cytidine. Accurate mass analysis confirmed the empirical
formulae of the ions at m/z 314 and 182 (Table 2(b)) as the
[MHþ] and [BHþ] of ethylacetylcytidine.
Peak 8 (and second ion found in peak 9)Peak 8 has a retention time of 28.5 min and its spectrum
shows an ion at m/z 271. Fragmentation of this ion gives a
single product ion at m/z 139 which loses water upon
further fragmentation in the ion trap to give m/z 121 (data
not shown). The loss of 132 Da between the ions at m/z 271
and 139 is considered to represent loss of a ribose sugar, and
is indicated in the 132 Da CNL spectrum (Fig. 12) (given that
the structure is a nucleoside or similar compound). The
only compound matching the data given is a pyridine-
carboxamide-one riboside (PCNR). The present data does
not allow us to distinguish which possible isomer (of which
Rel
ativ
e A
bund
ance
m/z
80 100 120 140 160 180 200 220 240 260 280 3000
10
20
30
40
50
60
70
80
90
100168.0
300.0
95.1 112.2
(b)
Rel
ativ
e A
bund
ance
m/z
100 200 300 400 500 600 700 8000
10
20
30
40
50
60
70
80
90
100 300.1
(a)
Figure 8. MS analysis of peak 3: (a) full-scan ion trap mass spectrum; (b) MS/MS ofm/z 300; (c) CNL
of 132Da, scanned between m/z 299.5 and 300.5; (d) MS3 of m/z 300! 168; and (e) MS4 of m/z
300! 168!150.
146 A. Bond et al.
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 137–150
Rel
ativ
e A
bund
ance
Rel
ativ
e A
bund
ance
m/z
50 60 70 80 90 100 110 120 130 140 150 160 170 1800
10
20
30
40
50
60
70
80
90
100150.0
95.1168.0
112.1
m/z
80 90 100 110 120 130 140 1500
10
20
30
40
50
60
70
80
90
10095.0
150.0
(d)
(e)
Figure 8. Continued.
MS analysis of urinary nucleosides. V: pyrimidine nucleosides 147
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 137–150
two have been shown to exist naturally) it might be and so
the identification is given here as PCNR. A second ion with
an identical m/z value and MS/MS fragmentation profile
was also discovered in peak 9 (co-eluting with the acetylcy-
tidine and having a comparatively low abundance com-
pared with peak 8) when studied as part of the accurate
mass analysis. It is known that two isomers exist, as men-
tioned previously. Both these isomers were isolated and
identified by matrix-assisted laser desorption/ionisation
time-of-flight (MALDI-ToF) analysis recently,26 and are
N
NHCOCH2CH3
ON
O
OHOH
HH
HH
HO
N
NH
NHCOCH2CH3
O
N
NH
NH2
O
H+
H+
H+
m/z = 300
m/z 168 m/z 112
m/z 150
m/z 95
C
NH
C
N
O
+
-H2O
Figure 9. Fragmentation of peak 3, protonated methylacetylcytidine.
Figure 10. MS analysis of peak 6: (a) full-scan ion trap mass spectrum; (b) CNL of 132Da, scanned between m/z 313.5 and
314.5; and (c) CNL of 132Da, scanned between m/z 285.5 and 286.5.
148 A. Bond et al.
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 137–150
also thought to be detected here; however, the second lower
level isomer was only detected by the accurate mass analy-
sis. The accurate mass data obtained added further weight
to the identifications shown in Table 2(b) and interestingly
indicated a difference in MS/MS spectra generated by the
ion trap versus the Q-ToF. The ion trap product ion spectra
gave only the m/z 139 product ion, which upon further frag-
mentation lost a water molecule to generate a MS3 product
ion atm/z 121. The MS/MS analysis performed as part of the
accurate mass experiment generated the m/z 139 product
ion as the most abundant product ion, but also generated
a second product ion directly from m/z 271 at m/z 122. The
accurate mass derived empirical formula of this second
product ion matched that which would be expected by the
loss of both the ribose sugar and an NH3 from the precursor
ion. It is suspected that the difference seen arises due to dif-
ferent collision energies that can be applied by the two dif-
ferent types of mass spectrometer.
An overview of the identified pyrimidine nucleosides for
which no standards were available is given in Table 2.
CONCLUSIONS
Pyrimidine nucleosides were purified from urine and ana-
lysed by a variety of mass spectrometric methods in order
to identify those detected. For a number of the nucleosides
the initial mass spectrometric data suggested modified and
unmodified nucleosides of which commercial standards
were easily available. This allowed the comparative product
ion data, accurate mass analysis and retention time to be
studied in order to confirm the identity of these nucleosides.
The nucleosides identified by comparison with standard
compounds were the unmodified cytidine, cytidine with
acetyl and methyl modifications, and pseudouridine, the iso-
form of uridine. For the remaining suspected nucleosides no
commercial standards were available for comparison. The
identification of these nucleosides therefore relied on the
potential of the ion trap to perform not only MS/MS analysis,
but also further MSn fragmentation, providing more structur-
al information, the constant neutral loss scans suggesting the
modification state of the ribose sugar and the empirical for-
mulae obtained by accurate mass measurements (Q-TOF).
Additionally, the knowledge that the purification protocol
developed relies on the presence of a cis-diol group being pre-
sent in the ribose sugar moiety of the nucleoside assisted in
identifying these unknown nucleosides. Any loss of this cis-
diol group due to modification at either C2 or C3 of the ribose
would result in the nucleoside being lost during clean-up and
so these positions must remain modification free in order to
be purified by the applied protocol. Also of use was the
knowledge that initial fragmentation of nucleosides com-
monly leads to the cleavage of the glycosidic bond that joins
the ribose sugar and the nucleobase. Under mild MS/MS con-
ditions the simultaneous cleavage of the glycosidic bond
together with the cleavage of any modifications of the base
is never observed and so it was adopted by us that any
MS/MS analysis of a modified nucleoside producing an
unmodified base as the product ion must therefore be modi-
fied on the ribose sugar. Finally, the modifications were all
relatively small in nature, with addition of methyl groups
being common, and so the number of possibilities for the ten-
tative structures was limited.
Of specific interest were peaks 3, 6 and 9 which have been
identified as acetylcytidine and two novel nucleosides—
methylacetylcytidine and ethylacetylcytidine. A number of
other novel modified cytidine nucleosides were tentatively
identified in the current work and these included cytidine
with a formyl group on the 50-O position of the ribose ring and
a 50-deoxycytidine, as previously reported and interestingly
being thus far unique to the urine of patients with cancers of
the head and neck.9 In conclusion we feel that the identifica-
tions are conclusive enough to justify their acceptance due to
the informative fragmentation data and known behaviour of
the nucleosides under such conditions despite the absence of
commercial standards for comparison in some cases.
AcknowledgementsThis work was financially supported by the Evan Davies
Trust Fund and the Flemish Government GOA Action
(FWO-Vlaanderen 1.5.139.00 and G.2133.94).
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