Tandem Mass Spec ome ic S udy o Cip o loxacin-Poly(e hylene glycol)
Conjuga e in he P esence o Alkali Me al Ions
Sándo Kéki1, Lajos Nagy1, Ákos Kuki1, Gábo Pin é 2, Pál He czegh2, Miklós Zsuga1*
1Depa men o Applied Chemis y, Uni e si y o Deb ecen, H-4012 Deb ecen, Hunga y
2Depa men o Pha maceu ical Chemis y, Uni e si y o Deb ecen, H-4012 Deb ecen,
Hunga y
Abs ac
The agmen a ion and agmen a ion beha io s o singly, doubly, and iply cha ged adduc s
o cip o loxacin-poly(e hylene glycol) conjuga e (P_C ) wi h alkali me al ions, including Li+,
Na+ and K+ ions, gene a ed by elec osp ay (ESI) we e s udied as a unc ion o collision
ene gy. The p oduc ion spec a o adduc s wi h cha ge s a es +1, +2, and +3 a e domina ed
by p oduc ions a ising om he loss-neu al moie y (cip o loxacin) and CO2, and ions o med
by dissocia ion o he p ecu so ion ([P_C +xM]x+) in o p oduc ions [P+(x-1)M](x-1)+ and
[C +M]+ (whe e P_C , P and C ep esen he cip o loxacin-poly(e hylene glycol) conjuga e,
he poly(e hylene glycol) backbone wi hou he endg oups, and he cip o loxacin moie y,
espec i ely; M is he alkali me al ion and x is he cha ge). I was ound ha he me al ions do
no signi ican ly al e he agmen a ion pa e n o cip o loxacin-poly(e hylene glycol)
conjuga e. I is also in e es ing ha he un and he shape o he su i al yield cu es o he
singly and doubly cha ged adduc ions a e independen o he ca ion. Howe e , in he case o
iply cha ged adduc s, su i al yield cu es ollow each o he in he o de K+, Na+, and Li+.
Based on he expe imen al esul s, a agmen a ion mechanism o he singly and mul iply
cha ged adduc s o P_C wi h alkali me al ions is gi en. In addi ion, a en a i e desc ip ion o
he signal in ensi y a ia ions o he p oduc ions wi h he lab ame collision ene gy is also
epo ed.
Keywo ds: cip o loxacin-poly(e hylene glycol) conjuga e, agmen a ion, ene gy- a iable
collision-induced dissocia ion, ca ion a achmen
1
In oduc ion
Cip o loxacin [1] is he mos p ominen membe o a g oup called he luo oquinolones.
Cip o loxacin is equen ly used as a wide-spec um an ibio ic o ea and p e en in ec ions
caused by bac e ia bo h in human and animal bodies. Addi ionally, cip o loxacin has also
been app o ed as a e y e ec i e medicine o coun e ing he inhaled o m o an h ax [2].
Due o he po en ial isk o es ablishing esis ance caused by an ibio ic esidues in oods o
animal o igin, he Eu opean Union Commi ee has se legisla ion o he le el o di e en
an ibio ics esiding in oods u s [3]. Pa ly owing o hese igo ous egula ions, se e al
me hods o he quali a i e and quan i a i e de e mina ion o an ibio ics has been applied o
hese pu poses; howe e , he mos selec i e o hese me hods is liquid ch oma og aphy (LC)
combined wi h mass spec ome y (MS) [4-10]. The e o e, he agmen a ion p ope ies o
a ious an ibio ics, including ha o cip o loxacin unde elec osp ay condi ions, ha e been
s udied [11,12].
Du ing ou esea ch on he modi ica ion o se e al an ibio ics, we aimed a he pegila ion o
cip o loxacin, i.e., he syn hesis o cip o loxacin-poly(e hylene glycol) conjuga e (P_C ).
Mass spec ome y analysis o P_C conjuga e showed ha unde elec osp ay condi ions, i
eadily o ms singly and mul iply cha ged adduc s wi h alkali me al ions due o he p esence
o he poly(e hylene glycol) backbone. F om a mass spec ome y poin o iew, i is
impo an o ga he in o ma ion as o how he ca ion and he cha ge s a e o adduc s, as well
as he collision ene gy, a ec he agmen a ion beha io s o such conjuga es. In his a icle,
we epo a de ailed ene gy- a iable collision-induced dissocia ion s udy o singly and
mul iply cha ged adduc s o P_C conjuga e wi h alkali me al ions (K+, Na+, Li+)
Expe imen al
Chemicals.
All chemicals used we e ecei ed om Ald ich (Seelze, Ge many). Sample solu ions we e
p epa ed in me hanol a a concen a ion o 1 mM cip o loxacin-poly(e hylene glycol)
conjuga e (P_C ) and 1 mM alkali me al chlo ides (LiCl, NaCl and KCl).
Syn hesis o he cip o loxacin-poly(e hylene glycol) conjuga e (P_C )
Syn hesis o he cip o loxacin-poly(e hylene glycol) conjuga e (P_C ) began om
poly(e hylene glycol) wi h a numbe a e age molecula weigh o 1000 g/mol possessing
-OCH3 and -OH endg oups. The –OH e mini we e con e ed in o -NH2 e mini, ollowed by
2
he la e ’s con e sion o iso hiocyana e and subsequen eac ion wi h cip o loxacin o gi e
cip o loxacin-poly(e hylene glycol) conjuga e. P_C was cha ac e ized by using 1H-NMR,
13C-NMR, and IR-spec oscopy. The de ails o he syn hesis will be epo ed in a sepa a e
pape . The chemical s uc u e o P_C is shown in Scheme 1.
Scheme 1.
Ins umen a ion.
Elec osp ay Quad upole Time-o -Fligh MS/MS (ESI-Qq-TOF).
MS/MS measu emen s we e pe o med wi h a Mic oTOF-Q ype Qq-TOF MS ins umen
equipped wi h an ESI sou ce om B uke (B uke Dal oniks, B emen, Ge many). The sample
solu ions we e in oduced di ec ly in o he ESI sou ce wi h a sy inge pump (Cole-Pa me Ins.
Co., Ve non Hills, IL, USA) a a low a e o 2 µL/min. The empe a u e o he d ying gas
(N2) was main ained a 100oC. The needle ol age was 4 kV. Fo MS/MS expe imen s,
ni ogen gas was used as he collision gas and he collision ene gies we e a ied in he ange
o 10-100 eV (in he labo a o y ame). The p essu e in he collision cell was de e mined o be
8x10-3 mba . The p ecu so ions o MS/MS we e selec ed wi h an isola ion wid h o 5 Th. All
he MS and MS/MS spec a we e accumula ed and eco ded by a digi ize a a sampling a e
o 2 GHz. The mass spec a we e calib a ed ex e nally using he exac masses o clus e s
[(NaTFA)n+Na]+ (n=1-3) gene a ed om he elec osp ayed solu ion o sodium
i luo oace a e (NaTFA). The accu acy o mass de e mina ion was wi hin +8 ppm. The mass
spec a eco ded we e e alua ed by he Da aAnalysis 3.1 so wa e om B uke .
Resul s and Discussion
ESI-TOF MS o cip o loxacin-poly(e hylene glycol) conjuga e
The elec osp ay mass spec a o cip o loxacin-poly(e hylene glycol) conjuga e (P_C ) in he
p esence o alkali me al ions (Li+, Na+, K+) e ealed he p esence o adduc s wi h alkali me al
ions o cha ge s a es anging om +1 o +4. Addi ionally, besides hese adduc ions (mos
p obably due o he basic cha ac e o N a oms in he cip o loxacin endg oup), p o ona ed
molecules o P_C ([P_C +H]+) and mixed adduc s e.g., [P_C +H+Na]2+ wi h ela i e low
in ensi y also occu ed in he ESI-MS spec a. As a ep esen a i e example, he ESI-MS
spec um o P_C in he p esence o sodium ions is p esen ed in Fig. 1.
3
Fig. 1.
As is e iden in Fig. 1, unde elec osp ay condi ions, he o ma ion o adduc s [P_C +xNa]x+
wi h x=1 o 4 a e p e e ed wi h he p esence o some low-in ensi y mixed adduc ions, e.g.,
[P_C +H+Na]2+. Simila mass spec a we e eco ded o he elec ospayed solu ion o P_C
con aining Li+ and K+ ions. Howe e , i was ound ha he deg ee o polyme iza ion (DP,
i.e., numbe o epea uni s) belonging o he mos p obable peak a ied wi h cha ge s a e and
he ca ion, as depic ed in Fig. 2.
Fig. 2.
As shown in Fig. 2, he DP o he mos p obable peak inc eases wi h he cha ge s a e. This
e ec is p esumably due o he ac ha longe chains can accommoda e mo e ions owing o
coulombic epulsion, he eby shi ing he mos p obable peak posi ion o a highe DP alue.
On he o he hand, he ca ion also a ec s he DP o he mos p obable peak. I is also e iden
om Fig. 2 ha he la ge he ca ion size, he highe he DP alue o he mos p obable peak
o each cha ge s a e, as judged om he esul s ob ained o Li+ and Na+. (The ionic adii o
Li+ and Na+ a e 80 and 120 pm, espec i ely.) Howe e , he esul o K+ does no i in he
abo e end, since he ionic adii o K+ (180 pm) a e highe han hose o Li+ and Na+, and he
obse ed DP alue lies be ween he wo. The eason o his may be a mo e speci ic
in e ac ion o he K+ ions no only wi h he oxygen a oms o e hylene oxide epea uni s, bu
also wi h hose o he endg oups (cip o loxacin moie ies). The ca boxyl g oup in he
cip o loxacin moie y may enable he o ma ion o a sal b idge wi h he alkali me al ions.
Howe e , he o ma ion o such s uc u e is mos p obable wi h po assium ion and a sal
b idge wi h his ion may dec ease he size o PEG chain equi ed o liga e mos a o ably wo
K+ ions as sugges ed by one o ou e iewe s. The o ma ion o such sal b idge may be he
eason o he di e en end obse ed o K+ ion.
ESI-TOF MS/MS o cip o loxacin-poly(e hylene glycol) conjuga e
To s udy he agmen a ion p ope ies o P_C ca ionized wi h alkali me al ions, some
oligome peaks we e selec ed o MS/MS expe imen s. The p ima y goal o he p esen s udy
was o in es iga e he e ec s o he ca ions on he agmen a ion p ope ies o P_C as a
unc ion o he collision ene gy. The ene gy ans e ed o he p ecu so ion in collisions wi h
4
he backg ound gas molecules depends on he mass and he deg ee o eedom o he
p ecu so ion. The e o e, oligome peaks wi h app oxima ely he same numbe o epea uni s
we e selec ed o adduc s o each ca ion in he MS/MS expe imen s, allowing di ec
compa ison o he e ec o ca ions on he agmen a ion beha io wi hou any u he
co ec ions. (In his case, he molecula weigh and he deg ees o eedom (DOF) o he
p ecu so ion we e p ac ically cons an , o he wise he ob ained da a would need o be
co ec ed wi h he mass and DOF alues.) Fo his eason, P_C wi h epea uni numbe s o
21-22, 24-25, and 27-28 we e selec ed o MS/MS expe imen s in he case o singly, doubly,
and iply cha ged adduc s, espec i ely. A ep esen a i e i s -gene a ion p oduc ion
spec um (ESI-QqTOF MS/MS) o he p ecu so ion [P_C +2Na]2+ is shown in Fig. 3.
Fig. 3.
As can be seen in Fig 3, he p ecu so ion [P_C +2Na]2+ dissocia es o gi e p oduc ion
[P+2Na]2+ by he loss o a neu al cip o loxacin moie y. In pa allel, elimina ion o a CO2
molecule om he p ecu so also akes place, yielding p oduc ion [P_C –CO2+2Na]2+. The
elimina ion o CO2 om [P_C +2Na]2+ indica e ha a sal b idge wi h Na+ ion was also
o med o some ex en as sugges ed by one o ou e iewe s. The p esence o [C +Na]+ ion
indica es ha he cha ge is e ained on he cip o loxacin moie y. A singly cha ged p oduc ion
such as [P+Na]+ was also o med. The b eakdown cu es o he p ecu so ion [P_C +2Na]2+
a e p esen ed in Fig. 4.
Fig. 4.
As seen in Fig. 4, he in ensi y o [P+2Na]2+ p oduc ion inc eases apidly in he ange om
30 eV o 50 eV. The pa allel occu ence o he p oduc ion [P+Na]+ wi h ha o [C +Na]+
sugges s ha he o me was p oduced by he p ocess [P_C +2Na]2+  [P+Na]+ + [C +Na]+
a he han by [P+2Na]2+ [P+Na]+ + Na+. A highe collision ene gies, especially abo e 60
eV, he in ensi y o [P+2Na]2+ p oduc ion dec eases, mos p obably due o dissocia ion in o
[P+Na]+ and Na+. Deple ion o all o he singly cha ged p oduc ions wi h he inc easing
collision ene gy is a consequence o de achmen o sodium ion om he co esponding ion o
yield neu al molecules and ba e sodium ions. B eakdown cu es o he iply cha ged P_C
shows cha ac e is ics (Fig. 5) simila o hose o he doubly cha ged ions, wi h some
excep ions.
5

Fig. 5.
P oduc ions o med by loss o a CO2 molecule om he p ecu so appea ed only wi h e y
low in ensi y. In con as , he majo p oduc ion [P+2Na]2+ was p obably o med by he
dissocia ion o [P_C +3Na]3+ in o ions [P+2Na]2+ and [C +Na]+.
The collision-induced dissocia ion o singly cha ged sodia ed adduc s o P_C gi e only wo
p oduc ions, [P_C –CO2+Na]+ and [P+Na]+. I is in e es ing o no e ha o ma ion o he
p oduc ion [C +Na]+ was no obse ed a all. B eakdown cu es o he singly cha ged
sodia ed adduc (no shown) e ealed ha he in ensi y o [P+Na]+ inc eased wi h he collision
ene gy up o 80 eV, bu in he ange o 80-100 eV (in he labo a o y ame) he in ensi y o
[P+Na]+ emained p ac ically cons an . This obse a ion indica es ha he adduc ion [P+Na]+
is s able enough o su i e collisions o such high ene gy. Thus, he conclusions d awn o he
singly, doubly, and iple cha ged sodia ed adduc s o P_C a e shown o also hold o he
co esponding adduc s o P_C wi h Li+ and K+ ions.
The su i al yield (φ) was calcula ed using Eqn. 1, and he plo s o
φ
as a unc ion o he
collision ene gy o each cha ge s a e and ca ion adduc a e shown in Fig. 6.
)/(
ip ecu so p ecu so
III
Σ+=
φ
(1)
whe e Ip ecu so is he in ensi y o he p ecu so ion and ΣIi is he sum o he in ensi ies o all
p oduc ions appea ing in he spec um.
Fig. 6.
As e iden in Fig. 6, he un and he shape o he su i al yield cu es o he singly and
doubly cha ged adduc ions a e independen o he ca ion. Howe e , in he case o iply
cha ged adduc s, su i al yield cu es ollow each o he in he o de K+, Na+, and Li+,
indica ing ha po assia ed adduc s dissocia e mo e easily han li hia ed adduc s do. These
obse a ions may indica e ha he e ec o cha ge epulsion be ween he ca ions on he
dissocia ion o adduc s becomes mo e p onounced o iply cha ged adduc ions.
6
The main agmen a ion schemes p oposed o he singly and mul iply cha ged adduc s o
P_C wi h alkali me al ions a e summa ized in Scheme 2.
Scheme 2.
Acco ding o Scheme 2, he dissocia ion o he p ecu so ion [P_C +xM]x+ akes place
simul aneously ia p ocesses deno ed by a, b, and c. In p ocess a, he loss o an endg oup is
ope a ional, yielding p oduc ions [P+xM]x+ and a neu al agmen C . Fu he decomposi ion
o [P+xM]x+ can also ake place by de achmen o he M+ ion o gi e ion
[P+(x-1)M](x-1)+ as indica ed (p ocess d). P ocess b yields p oduc ions [P+(x-1)M](x-1)+ and [C
+M]+. No e ha he la e p ocess occu s in he case o doubly and iply cha ged adduc s,
when x = 2 and 3. A simila p ocess does no ake place o he singly cha ged adduc , as no
[C +M]+ ions in he p oduc ion spec a o [P_C +M]+ could be de ec ed. Pa allel o p ocesses
a and b, elimina ion o a CO2 molecule om he p ecu so ion also ope a es (p ocess c),
gi ing p oduc ion [P_C –CO2+xM]x+ which may unde go u he decomposi ion o yield
ions [P+(x-1)M](x-1)+ and [C –CO2+M]+ (p ocess e). I should be emphasized, howe e , ha
ions [P+(x-1)M](x-1)+ and [C –CO2+M]+ occu only wi h e y low in ensi ies as compa ed o
hose o he o he p oduc ions. On he o he hand, o ma ion o p oduc ion [P_C –
CO2+xM]x+ in he case o iply cha ged adduc s (x = 3) can be de ec ed only wi h e y low
in ensi y. Simple de achmen o M+ om he p ecu so ion [P_C +xM]x+ o gi e ion [P_C +
(x-1)M](x-1)+ occu ed a a de ec able le el only in he case o x = 3 (see Fig. 5); he e o e, his
p ocess was no conside ed in Scheme 2.
Kine ic conside a ion o p oduc ion o ma ion and deple ion
In gene al, in he case o pa allel, consecu i e eac ions when he ini ial eac an decomposes
by se e al pa allel i s -o de eac ions, each wi h a e cons an o ki, and he pa icula
in e media e deno ed by P1 u he decomposes wi h a e cons an k1’ in a i s -o de eac ion,
hen he concen a ion o P1 (CB) as a unc ion o he eac ion ime (
τ
) can be gi en by
)]exp()[exp(
,
1
,
1
,
,
1
1
ττ
kk
kk
ck
c
i
i
oA
P
−−Σ−
Σ−
=
(2)
7
whe e CA,o is he ini ial concen a ion, and Σki is he sum o he a e cons an s o pa allel
eac ions.
In andem mass spec ome y expe imen s, he “ eac ion ime” is p ac ically cons an while
he in e nal ene gy gained du ing collisions is a ied. As a i s app oxima ion, he
dependence o he a e cons an (k) on he e ec i e empe a u e (Te ) is assumed o obey he
A henius equa ion as:
)exp(
e
a
RT
E
Ak
−=
(3)
Te is no a eal empe a u e, since he sys em is no in a he mal equilib ium; howe e , he
alue o Te can be app oxima ed by Eqn. 4,
R
EE
T
o
e
ν
in in ,
+
=
(4)
whe e ν and R a e he numbe o deg ees o eedom and he gas cons an , espec i ely, and
Ein ,o and Ein ep esen he ini ial in e nal ene gy o he p ecu so ion and he in e nal ene gy
acqui ed du ing collisions, espec i ely.
The in e nal ene gy gained du ing collision is p opo ional o he labo a o y ame collisional
ene gy (Ec), as shown in Eqn. 5.
c
EE
α
=
in
(5)
whe e α is he p opo ionali y cons an , which depends on se e al ac o s such as he c oss-
sec ions, he molecula mass, and he e iciency o ene gy ans e om he backg ound gas
molecules [13,14].
Subs i u ing Eqn. 5 in o Eqn. 4 hen in o Eqn. 3, and assuming ha Ein ,o << Ein , he
dependence o an unimolecula a e cons an on Ec can be gi en by Eqn. 6.
)/exp(
c
EbAk
−=
(6)
whe e
αν
/
a
Eb
=
.
8
Assuming linea ela ionships be ween he signal in ensi ies and he gas-phase concen a ions
o he co esponding ions,
11
PP
cI
β
=
(7)
whe e
1
P
I
and
1
P
c
a e he in ensi y and he concen a ion o P1 ion, and β is he
p opo ionali y cons an .
Subs i u ing Eqn. 2 in o Eqn. 7 wi h ea angemen s esul s in he dependence o signal
in ensi y o P1 on he labo a o y ame collision ene gy as gi en by
)]exp()[exp(
,
1
,
1
,
,
1
1
ττ
ττ
βτ
kk
kk
ck
I
i
i
oA
P
−−Σ−
Σ−
=
(8)
Using pa ame e s a1, a2, a3, b1, and b2 o he ollowing exp essions,
)/exp(
11
,
1
c
Ebak
−=
τ
,
)/exp(
22
ci
Ebak
−=Σ
τ
, and
3,
ac
oA
=
β
, and by subs i u ion o hese exp essions in o Eqn.
8, he heo e ical dependence o he signal in ensi y on he lab ame collision ene gy can be
simula ed o i he expe imen al in ensi ies. Fo he sake o simplici y, we assume a single
exponen ial exp ession o
τ
i
k
Σ
. Figu e 7 shows he dependencies o he signal in ensi ies
on he lab ame collision ene gy (Ec) o he p oduc ion [P+2Na]2+ o med om he
p ecu so ions [P_C +2Na]2+ and [P_C +3Na]3+, oge he wi h he cu es calcula ed by Eqn. 8.
The alues o Ec e sus he in ensi ies o [P+2Na]2+ ions a e lis ed in Table 1.
Fig. 7.
Table 1.
As i u ns ou om Fig. 7, he expe imen al in ensi ies o he p oduc ions can be desc ibed
using he kine ic app oxima ions depic ed abo e. Mo eo e , a mo e p ecise desc ip ion o he
p ocesses occu ing in a mass spec ome e is also possible by means o a highly sophis ica ed
compu e p og am based on he Rice-Ramspe ge -Kassel-Ma cus (RRKM) heo y [14].
9
Scheme 3.
A
P
1
P
2
P
1
'
P
2
'
P
i
'
P
i
k
1
k
2
k
i
k
1
'
k
2
'
k
i
'
16

Fig. 1.
0.00
0.25
0.50
0.75
1.00
1.25
4
x10
In ensi y
400 600 800 1000 1200 1400 1600 m/z
+1
+2
+3
17
Fig. 2.
17
19
21
23
25
27
29
0 1 2 3
cha ge
numbe o epea uni s
Li
Na
K
18
Fig. 3.
0
1000
2000
3000
4000
5000
In ensi y
200 400 600 800 1000 1200 m/z
[C +Na]
+
[P+Na]
+
[P_C +2Na]
(p ecu so ion)
2+
[P+2Na]
2+
[P_C +2Na]
2+
-CO
2
19
Fig. 4.
0
1000
2000
3000
4000
5000
6000
7000
10 30 50 70 90
E
c
(eV)
In ensi y
[P+Na]+
[P_C +2Na]2+
[P_C -CO2+2Na]2+
[P+2Na]2+
[C +Na]+
20
Fig. 5.
0
500
1000
1500
2000
2500
15 25 35 45 55
E
c
(eV)
In ensi y
[C +Na]+
[P+3Na]3+
[P_C +3Na]3+
[P+2Na]2+
[P_C +2Na]2+
21

Fig. 6.
0
0.2
0.4
0.6
0.8
1
0 20 40 60 80 100
E
c
(eV)
φ
Li+
Na+
K+
+1
+2
+3
22
Fig. 7.
0
1000
2000
3000
4000
5000
6000
7000
10 30 50 70 90
E
c
(eV)
In ensi y
[P+2Na]2+ ([P_C +3Na]3+)
[P+2Na]2+ ([P_C +2Na]2+)
23