analysis of urinary nucleosides. v. identification of urinary pyrimidine nucleosides by liquid...

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


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.


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



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.


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.



Figure 6. MS analysis of peak 7: (a) full-scan ion trap mass spectrum; (b) MS/MS of m/z 272; and (c) CNL


144 A. Bond et al.


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.



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.


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.



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.


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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analysis of urinary nucleosides. v. identification of urinary pyrimidine nucleosides by liquid...

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