Qu
uantitattion of
o Nucleotides an
nd Nu
ucleosiides w
withoutt
Capilla
de
erivatiza
ation using
u
ary Ele
ectroph
horesis Coup
pled to
o
Ma
ass Spectrom
metry (C
CESI-M
MS)
CES
SI-MS Metho
od for Analys
sis of Challen
nging Small Molecule M
Metabolites a
and Similar D
Drug Produccts
Jose
e-Luis Galleg
gos-Perez
SCIE
EX Separatio
ons, Framing
gham, MA
Intro
oduction
CESI-MS is the inte
egration of capillary electroph
horesis (CE) with
electrrospray ionization (ESI), intto a single dynamic process
within
n the same de
evice (Figure 1).
1 Operating at ultra-low flo
ow
rates,, it helps reduce ion supp
pression and increases
i
assay
sensittivity for op
ptimal results.. Nucleotides
s are charge
ed
moleccules, precursors and break
k-down produc
cts of DNA an
nd
RNA that also form the basic structure of cofacttors. They act as
a
gy carriers and
d mediators of important cellular processes
s1 .
energ
Nucle
eosides are ne
eutral and are also vital cellu
ular componen
nts
that inside the cell are metaboliz
zed by nucleokinases into th
he
2
e nucleotide mono-, di-, triphosphates
s . Nucleoside
es
active
analo
ogues are use
ed as therapeu
utics of the trreatment of virral
infecttions, such as the human im
mmunodeficien
ncy virus (HIV
V)1.
These
e polar comp
pounds are us
sually difficult to analyze by
traditiional chromatography tech
hniques, nee
eding an exttra
deriva
atization step. This technicall note describe
es an alternativ
ve
workfflow using CES
SI-MS for the quantitative ana
alysis of selecte
ed
nucle
eotides and nucleosides without derivatization with
reproducibility and detection at nM levels. Ap
pplication of th
his
i promising for analysis off these types of
CESI-MS method is
e pharmaceu
utical and biological (e..g.
moleccules in the
metab
bolomics, neurrosciences) fields among othe
ers.
Figure
e 1. CESI-MS: CE integrated
d with QTRAP®
® 6500 MS
erimental P
Procedure
Expe
Reage
ents:

Adv
vantages of CESI-MS Method

No derivatiization step

Short analy
ysis time

Sensitive, quantitative
q
me
ethod
Ammonium acetate (AA), formic acid (F
FA) and, acetic
om Sigma-Aldrich and used
d
acid were purchased fro
ation. Ultrapure
e water (Type 1)
without any further purifica
ed from a Miilli-Q Direct U
Ultrapure Wate
er
was obtaine
System. cyytidine 5′-tripho
osphate (CTP
P), cytidine 5′diphosphate
e (CDP), cytidin
ne 5'-monopho
osphate (CMP)),
adenosine ttriphosphate (A
ATP), guanosin
ne triphosphate
e
(GTP),
urridine
triphossphate
(UTP
P),
thymidine
triphosphate
e (TTP), rridine
e (Ur) and thym
midine (Th) were
e
purchased ffrom Sigma-Ald
drich.
Samplle Preparation
n:

ons of standarrd compounds were prepared
Stock solutio
d
in DDI wate
er. Standard so
olutions, conta
aining a mixture
e
of the analyytes, in the ran
nge of 0.25, 0.5
50, 1, 5, 10, 25
and 50 nM w
were also prepared in DDI wa
ater.
p1
Instru
umentation:

Capillary Electrophore
esis (CE): Se
eparations we
ere
he
performed using a CESI 8000 Plus frrom SCIEX. Th
ed using 32 Ka
arat™ softwa
are
instrument was controlle
v10.

Capillary: A bare fused-s
silica capillary was
w used for th
he
separation; 30 m i.d. x 91
9 cm total leng
gth.

Mass Spe
ectrometry (M
MS): A QTRA
AP® 6500 mass
spectrometter (SCIEX) co
oupled to the CESI
C
through th
he
CESI-MS Sprayer
S
interfa
ace (see Figure
e 2) operating in
positive ion
n mode with An
nalyst 1.7® sofftware.
ment Conditio
ons:
Instrum

MS: The Q
QTRAP® 6500 mass spectro
ometer was run
with electro
ospray ionizatio
on in positive ion mode and
d
acquisition w
was from 100 tto 600 m/z valu
ues. Conditions
for Multiple Reaction Moniitoring (MRM) were evaluated
d
pound to selectt precursor and
d
individually for each comp
ons. Table 1 provides the transitions tha
at
fragment io
were used fo
or the determin
nation.
Table 1. MRM trans
sitions for the
e analyzed co
ompounds and
d
the low
west detected concentrations (nM) for ea
ach one.
Figurre 2: CESI Sprrayer Interface
e
Data A
Analysis

Instru
ument Conditiions:

CESI: Several backgro
ound electroly
ytes (BGE) are
a
recommended in the litterature1. Amm
monium aceta
ate
ve
was selectted because it was demonsttrated in positiv
ionization mode.
m
Severall experimental conditions we
ere
tested to achieve
a
the be
est separation using adenosin
ne
triphosphatte (ATP) as model
m
analyte. Formic acid wa
as
used as stacking age
ent. The fina
al experimenttal
conditions were:
pectra were ca
arried out using
g
Analyses off the mass sp
Peak View®
® software (SC
CIEX, Framingham, MA). The
quantitative data analyysis was performed using
g
MultiQuant™
™ software (SC
CIEX, Framingh
ham, MA).
Metho
od parameters: See Figures 3 and 4.
6
6
1% FA
ate
Background electrolyte: 12.5 mM Ammonium aceta
pH 9.7.
olution: 1% Formic acid
Stacking so
5
5
4
4
3
3
2
2
1
Separation
n voltage: 30 kV with 2 psi of assiste
ed
pressure
orage temperatture: 10 ºC
Sample sto
Capillary te
emperature: 20
0 ºC
BGE
A
1
BGE
B
H2O
HCl
NaOH
C
D
E
RAY
INLET BUFFER TR
BGE
F
A
H2O
B
E
C
D
OUTLET BUFFFER TRAY
F
BGE: 12.5 mM Ammonium accetatte, pH 9.7
e 3: Scheme o
of the inlet and outlet buffe
er vial tray settFigure
up for the CESI sep aration.
p2
Figurre 4: Separatio
on Method Parameters. In step
s
10 and 12
2, use 2 psi off forward pres
ssure. In steps
s 3, 5, 6 and 13 a low
separration voltage
e is applied with 100 psi in both
b
directions to rinse in p
parallel the cap
pillary in the s
separation and
d conductive
lines.
p3
Res
sults and Discussion
D

Analytical parameters such as peak shape an
nd
t
were ev
valuated by in
njecting sample
es
migration times
individually
y. A standard mixture was also injected to
check sepa
aration perform
mance (Figure 5).
5

All compou
unds showed migration time
es below 15 min
m
with high re
eproducibility and
a sharp peak
k shapes (Figu
ure
5).

Linearity and
a
other para
ameters were evaluated usin
ng
analyte sample mixtures
s from 0.25 – 50 nM for eac
ch
compound.

Figure 6 shows
s
extracte
ed electropherograms for th
he
lowest co
oncentration detected
d
for the individu
ual
compounds
s, ranging from
m 0.25 to 1 nM
M for nucleotide
es
and 5 nM for
f nucleosides
s (see Table 1).

Calibration curves were obtained usin
ng MultiQuant™
software (SCIEX,
(
Framingham, MA). The analytic
cal
method exhibited a quantitative linear re
esponse with r >
0.9 (Figure
e 7).
e 5: Extracted
d Ion Electro
opherograms (XIEs) of the
e
Figure
analyzzed compound
ds. All injected
d in the same experiment at
a
10 nM (see Table 1)..
Con
nclusions

A method for separatio
on of some nucleotides
n
an
nd
es was develop
ped for CESI-MS with a sho
ort
nucleoside
analysis tim
me and minimu
um required sa
ample volume. In
addition this separation does
d
not require derivatizatio
on
ytes.
of the analy

Separation
n of challenging charged mo
olecules: cytidin
ne
phosphates, ATP, GTP, UTP, TTP and nucleosides (Ur
h
been demonstrated usin
ng the CESI-M
MS
and Th) has
system.

CESI-MS could
c
be applie
ed for quantitattive analysis with
low detecte
ed concentratio
ons (< 5 nM) for the analyze
ed
compounds
s.

Application
n of this CESI method is
s promising for
f
metabolom
mics, biotransfformation pharmacology, an
nd
analytical
characterization
stu
udies
whe
ere
pe molecules are
a quantitative
ely
nucleotide//nucleoside-typ
analyzed, particularly wh
hen samples are obtained with
low volume
es and/or and concentrations.
c
.
e 6: Extracted Ion Electroph
herograms (XIE
Es) for each of
o
Figure
the a nalyzed com
mpounds at the lowest concentration
n
detectted.
p4
Thym
midine triphosphatte (TTP)
Uridin
ne (Uri)
Thym
midine (Thy)
Figurre 7: Calibration curves fo
or the analyze
ed compound
ds.
All an
nalyte concen
ntrations are ra
anging from 0 to 50 nM.
Refe
erences
1. Willlems, An V., Dieter
D
L. Deforc
ce, Carlos H. Van Peteghem,
and JJan F. Van Boc
cxlaer. "Analysis of nucleic acid constituents
by on
n‐line capillary electrophoresis
e
s‐mass spectro
ometry."
Electrrophoresis 26, no. 7‐8 (2005)): 1221-1253.
2. Liu
u, Charles C., et
e al. "Capillary electrophoresiis‐electrospray
y‐
masss spectrometry of nucleosides
s and nucleotide
es: Application
to pho
osphorylation studies
s
of anti‐h
human immuno
odeficiency viru
us
nucle
eosides in a hum
man hepatoma
a cell line." Elec
ctrophoresis
26.7‐8
8 (2005): 1424
4-1431.
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earch Use Only. Nott for use in diagnostic procedures. AB Sc
ciex is doing businesss as SCIEX.
The trad
demarks mentioned herein are the prope
erty of AB Sciex Pte.. Ltd. or their respecttive owners. AB SCIIEX™ is being used under license.
Docume
ent number: RUO-M
MKT-02-2335-A
p5