Ci a ion: A enas, M.; Ma ín, J.;
San os, J.L.; Apa icio, I.;
Fe nández-San ancisco, O.; Alonso,
E. Compa ison o Di e en
Techniques o he De e mina ion o
Pla inized Cy os a ic D ugs in U ine
Samples. Molecules 2022,27, 8139.
h ps://doi.o g/10.3390/
molecules27238139
Academic Edi o : And eas Tsakalo
Recei ed: 13 Oc obe 2022
Accep ed: 20 No embe 2022
Published: 23 No embe 2022
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molecules
A icle
Compa ison o Di e en Techniques o he De e mina ion o
Pla inized Cy os a ic D ugs in U ine Samples
Ma ina A enas 1, Julia Ma ín1,* , Juan Luis San os 1, I ene Apa icio 1, Oma Fe nández-San ancisco 2,3
and Es eban Alonso 1
1
Depa amen o de Química Analí ica, Escuela Poli écnica Supe io , Uni e sidad de Se illa, C/Vi gen de Á ica 7,
E-41011 Se illa, Spain
2Depa amen o de Medicina P e en i a y Salud Pública, Facul ad de Fa macia, Uni e sidad de G anada,
E-18071 G anada, Spain
3A hisa Biogene ación, c/Pe iodis a F ancisco Ja ie Cobos, nº18, E-18014 G anada, Spain
*Co espondence: [email p o ec ed]
Abs ac :
Pla inum-based cy os a ic d ugs a e one o he mos widely used cance ea men s. They
a e exc e ed ia he u ina y ac and can each he en i onmen h ough was ewa e , posing a isk o
human heal h due o hei side e ec s. Fou iden i ica ion and quan i ica ion echniques, including
liquid ch oma og aphy (LC) sepa a ion coupled o (i) a diode a ay ul a iole (UV(DAD)) (ii), mass
spec ome e in single ion moni o ing mode (LC-MS) and (iii) mul iple eac ion moni o ing mode
(LC-MS/MS) and (i ) de i a iza ion wi h die hyldi hioca bama e p io o LC-MS/MS analysis, ha e
been op imized and compa ed o he mul i esidue de e mina ion o main pla inized cy os a ic d ugs
(cispla in, ca bopla in, and oxalipla in) in u ine samples. Pa ame e s ha a ec he e iciency o
he ch oma og aphic sepa a ion and analy ical de e mina ion o di e en me hods (column, mobile
phase, wa eleng h, p ecu so ions, agmen o , and p oduc ions) we e op imized. Analy ical ea u es,
such as ma ix e ec , sensi i i y, p ecision, selec i i y, and linea i y, we e calcula ed. In e ms o
selec i i y, he de i a iza ion echnique was disca ded since i was only applicable o he pla ina ed
sum. A high dilu ion o he sample wi h LC-UV(DAD) was needed o educe he ma ix e ec .
O e all, he LC-MS/MS me hod p esen ed he bes analy ical ea u es (% RSD
≤
12.8%, R
2≥
0.991,
o me hod-de ec ion limi s be ween 0.01–1
µ
g mL
−1
). The selec ed me hod was applied o he
quan i ica ion o pla inized cy os a ic d ugs in hospi al u ine samples om oncologic pa ien s.
Keywo ds:
cy os a ic d ugs; cispla in; ca bopla in; oxalipla in; LC-UV(DAD); LC-MS/MS; MRM
mode; SIM mode
1. In oduc ion
Cance anks as a leading cause o dea h and an impo an ba ie o inc easing li e
expec ancy in e e y coun y o he wo ld [
1
]. Las yea , he e we e an es ima ed nine een
million new cases a ound he wo ld, and mo e han hal o pa ien s e en ually die om
i (GLOBOCAN 2020 da abase). Cy os a ic d ugs a e equen ly used o ea cance and
non-neoplas ic illnesses [
2
]. Pla inized an ineoplas ic d ugs a e among he mos signi ican
an icance ea men s and a e success ully u ilized o ea a a ie y o human cance s such
as u ologic, gynecologic, pedia ic, es icula [
3
], lung, o a ian [
4
], and colon cance [
5
].
They can be used alone o in combina ion wi h o he cy os a ic d ugs. The mos popula
pla inum-based medica ions a e oxalipla in (
cyclohexane-1R,2R-diamine(oxala o)pla inum(II)),
ca bopla in (cis-diammine(cyclobu ane-1,1-dica boxyla e-O,O
0
)pla inum(II)), and cispla in
(cis-diamminedichlo opla inum(II)) [
3
]. Thei s uc u e consis s o a coo dina ion complex
con aining a pla inum a om in oxida ion s a e II (Figu e S1). These compounds in e e e
wi h he cell-di ision p ocess by in e ac ing wi h cell gene ical ma e ials. The mode o ac ion
consis s in ligands being eplaced by wa e molecules inside o he cells and p oducing
eac i e aqua ed complexes ha could di ec ly bind wi h DNA and inhibi cell di isions.
Molecules 2022,27, 8139. h ps://doi.o g/10.3390/molecules27238139 h ps://www.mdpi.com/jou nal/molecules
Molecules 2022,27, 8139 2 o 11
Howe e , his mechanism is non-selec i e and pla inized d ugs, as mos cy os a ic agen s,
may also a ec non- umo cells, which leads o geno oxic, mu agenic, and ca cinogenic
e ec s [
6
]. As a esul , conce n ega ding hei a e o use, hei elease in o he en i onmen ,
and hei possible haza dous impac s on he ecosys em and human heal h has g own. O
pa icula conce n a e he isks o heal hca e wo ke s, who a e exposed du ing d ug
p epa a ion, he ea men , and disposal o medicines, as well as h ough con ac wi h
pa ien exc emen . Recen wo ks ha e s udied he p esence o cispla in, among o he
cy os a ics, as a po en ial heal h haza d in ma ices p esen in hospi al en i onmen s, such
as ai , su aces, p o ec i e de ices, and equipmen [7–9].
Pla inized d ugs a e elimina ed ela i ely apidly h ough he pa ien ’s u ine [
6
].
Cispla in is exc e ed by his ou e by 28
±
4% wi hin 24 h o in a enous adminis a ion [
10
];
oxalipla in u ina y exc e ion is 53.8
±
9.1% 5 days a e adminis a ion [
11
] and ca bopla in
exc e ion a e is 93% du ing he i s 3 days [
12
]. Due o he high exc e ion a e, pa ien s’
u ine is he main ou e by which pla inized pollu an s each hospi als and municipal
was ewa e s [
6
], con amina ing su ace wa e , g ound, and c op soils o en i iga ed wi h
was ewa e e luen s o e ilized wi h sewage sludge o compos .
As he main ou e o en y o hese con aminan s in o he en i onmen , i is o in-
e es o de elop analy ical me hods o de e mine he p esence o hese pla inum-based
pollu an s bo h in biological and en i onmen al samples. Di e en analy ical me hods
ha e been p oposed o he indi idual de e mina ion o hese compounds [
13
] including
phospho escence in plasma and u ine [
14
], a omic abso p ion spec ome y in plasma and
mice o gans [
15
], liquid ch oma og aphy (LC) coupled o an ul a iole de ec o (LC-UV)
in blood [
16
] and in usion solu ions [
17
] o induc i ely coupled plasma mass spec ome y
(ICP-MS) in plasma [
18
] and hospi al was ewa e [
19
]. Howe e , hese echniques ha e
sho comings ega ding he mul i esidue de e mina ion, since hey ha e been applied o
he de e mina ion o a single cy os a ic (usually cispla in) o o al pla inum, sensi i i y, and
selec i i y. Mo e ecen ly, some LC me hods using andem mass spec ome y (MS/MS)
de e mina ion ha e been de eloped o analyze ca bopla in in plasma [
20
], ca bopla in and
oxalipla in in su aces [
21
] and cispla in, ca bopla in, and oxalipla in in su aces employing
zwi e ionic hyd ophilic in e ac ion liquid ch oma og aphy (HILIC) [
22
]. The e a e also
s udies ha ha e p oposed he de e mina ion o cispla in o ca bopla in indi ec ly h ough
de i a iza ion wi h die hyldi hioca bama e DDTC in plasma and u ine [
2
,
23
–
25
]. The e o e,
he aim o his s udy was o p opose an analy ical me hod o he simul aneous de e mina-
ion o cispla in, ca bopla in, and oxalipla in in human u ine. Fo ha , ou iden i ica ion
and quan i ica ion echniques, including liquid ch oma og aphy (LC) sepa a ion coupled
o (i) a diode a ay ul a iole de ec o (UV(DAD)) (ii), a mass spec ome e de ec o ope -
a ing in single ion moni o ing mode (LC-MS), and (iii) mul iple eac ion moni o ing mode
(LC-MS/MS) and (i ) de i a iza ion wi h die hyldi hioca bama e p e ious LC-MS/MS
analysis, ha e been compa ed and e alua ed.
2. Resul s
2.1. LC-UV(DAD) Me hod
To ob ain he highes sensi i i y and selec i i y o he ch oma og aphic sepa a ion in a
sho e un ime, he main a iables a ec ing ch oma og aphic sepa a ion and signal in en-
si y we e s udied. Fi s , a Zo bax Eclipse XDB–C18 Rapid Resolu ion HT (
50 ×4.6 mm i.d.
;
1.8
µ
m) and a HALO C-18 Rapid Resolu ion (50
×
4.6 mm i.d., 2.7
µ
m pa icle size) column
we e es ed in o de o ob ain a high sepa a ion pe o mance. The Zo bax Eclipse XDB–C18
Rapid Resolu ion column p o ided be e peak shape in he sho es ime wi h simila
esolu ion o all he s udied analy es.
The e ec o he mobile phase on ch oma og aphic sepa a ion was also s udied.
Me hanol (MeOH) and ace oni ile (AcN) we e e alua ed as o ganic modi ie s (
sol en B
).
As sol en A, deionized wa e wi h di e en addi i es was s udied. The selec ion o he
mobile phase was condi ioned by he de ec o s udied. Wi h UV-DAD, a good ch oma o-
g aphic sepa a ion is key o iden i y he analy es besides he UV spec um. The h ee
Molecules 2022,27, 8139 3 o 11
pla ina es we e sepa a ed in e e sed phase using micella liquid ch oma og aphy using
0.5 mM sodium dodecyl sulpha e (SDS) as he aqueous phase and MeOH as he o ganic
phase in isoc a ic mode (98:2, / ). The low a e was 0.6 mL min
−1
and he o al ch o-
ma og aphic un ime was 6 min.
The compounds we e iden i ied by compa ison o hei e en ion imes and UV spec a
wi h hose in he s anda d solu ion ch oma og ams. Peak a eas we e used o quan i ica ion.
Calib a ion cu es we e buil by linea eg ession o he peak a eas o he s anda d solu ions
agains hei concen a ions.
Pla inum d ugs we e measu ed a he maximum abso p ion wa eleng h, excep
ca bopla in, o which a comp omise had o be eached be ween he wa eleng h p o iding
he highes signal and he selec i i y o he peak. The UV de ec o was se a 210 nm o
cispla in and oxalipla in, and 254 nm o ca bopla in. Figu e 1shows he UV abso p ion
spec a o he pla inum d ugs s udied and he UV(DAD) abso p ion ch oma og am o
s anda d mix u e o he h ee pla inized d ugs unde he op imal condi ions. The peaks a e
well sepa a ed ( esolu ion 2.5 o cispla in-oxalipla in and 5.2 o oxalipla in-ca bopla in).
Molecules 2022, 27, x FOR PEER REVIEW 3 o 11
The e ec o he mobile phase on ch oma og aphic sepa a ion was also s udied.
Me hanol (MeOH) and ace oni ile (AcN) we e e alua ed as o ganic modi ie s (sol en
B). As sol en A, deionized wa e wi h di e en addi i es was s udied. The selec ion o
he mobile phase was condi ioned by he de ec o s udied. Wi h UV-DAD, a good ch o-
ma og aphic sepa a ion is key o iden i y he analy es besides he UV spec um. The h ee
pla ina es we e sepa a ed in e e sed phase using micella liquid ch oma og aphy using
0.5 mM sodium dodecyl sulpha e (SDS) as he aqueous phase and MeOH as he o ganic
phase in isoc a ic mode (98:2, / ). The low a e was 0.6 mL min−1 and he o al ch oma-
og aphic un ime was 6 min.
The compounds we e iden i ied by compa ison o hei e en ion imes and UV spec-
a wi h hose in he s anda d solu ion ch oma og ams. Peak a eas we e used o quan i-
ica ion. Calib a ion cu es we e buil by linea eg ession o he peak a eas o he s and-
a d solu ions agains hei concen a ions.
Pla inum d ugs we e measu ed a he maximum abso p ion wa eleng h, excep ca -
bopla in, o which a comp omise had o be eached be ween he wa eleng h p o iding
he highes signal and he selec i i y o he peak. The UV de ec o was se a 210 nm o
cispla in and oxalipla in, and 254 nm o ca bopla in. Figu e 1 shows he UV abso p ion
spec a o he pla inum d ugs s udied and he UV(DAD) abso p ion ch oma og am o
s anda d mix u e o he h ee pla inized d ugs unde he op imal condi ions. The peaks
a e well sepa a ed ( esolu ion 2.5 o cispla in-oxalipla in and 5.2 o oxalipla in-ca -
bopla in).
Figu e 1. (a) Abso p ion spec a o cispla in, oxalipla in, and ca bopla in; (b) Diode-a ay UV ab-
so p ion ch oma og am o a s anda d mix u e con aining 1 µg mL−1 o each pla inized d ug.
Figu e 1.
(
a
) Abso p ion spec a o cispla in, oxalipla in, and ca bopla in; (
b
) Diode-a ay UV
abso p ion ch oma og am o a s anda d mix u e con aining 1 µg mL−1o each pla inized d ug.
2.2. LC-MS Me hods
2.2.1. SIM Mode
The use o o mic acid, ace ic acid, ammonium o ma e, ammonium ace a e and
mix u es o hese acids and he co esponding sal s we e assayed as addi i es in he mobile
phase o imp o e he ioniza ion o he a ge compounds and hus sensi i i y. The pla inized
cy os a ic d ugs showed highe esponses wi h be e peak shapes using an isoc a ic elu ion
wi h an aqueous bu e solu ion con aining 20 mM ammonium o ma e (pH adjus ed o
6.4 using o mic acid) (95%) (A) and AcN (5%) as he mobile phase. The low a e was
0.6 mL min−1
and he injec ion olume was 10
µ
L. Analysis was pe o med in 5 min. The
Molecules 2022,27, 8139 4 o 11
MS de ec o was ope a ed in SIM mode, whe e a p ecu so ion is sough o iden i y and
de e mine he a ge compounds. The h ee pla inized compounds we e ionized in posi i e
mode, wi h [M + NH
4
]
+
being he mos abundan molecula ion o cispla in (318 m/z)
and [M + H]
+
o oxalipla in (398 m/z) and ca bopla in (372 m/z) (Figu e S3). O e all, he
abundance o he p ecu so ion o cispla in is lowe han o ca bopla in and oxalipla in
p ecu so ions. Figu e 2shows he SIM ch oma og am o a s anda d mix u e o he h ee
pla inized d ugs unde he op imal condi ions.
Molecules 2022, 27, x FOR PEER REVIEW 4 o 11
2.2. LC-MS Me hods
2.2.1. SIM Mode
The use o o mic acid, ace ic acid, ammonium o ma e, ammonium ace a e and mix-
u es o hese acids and he co esponding sal s we e assayed as addi i es in he mobile
phase o imp o e he ioniza ion o he a ge compounds and hus sensi i i y. The pla i-
nized cy os a ic d ugs showed highe esponses wi h be e peak shapes using an isoc a ic
elu ion wi h an aqueous bu e solu ion con aining 20 mM ammonium o ma e (pH ad-
jus ed o 6.4 using o mic acid) (95%) (A) and AcN (5%) as he mobile phase. The low a e
was 0.6 mL min−1 and he injec ion olume was 10 µL. Analysis was pe o med in 5 min.
The MS de ec o was ope a ed in SIM mode, whe e a p ecu so ion is sough o iden i y
and de e mine he a ge compounds. The h ee pla inized compounds we e ionized in
posi i e mode, wi h [M + NH4]+ being he mos abundan molecula ion o cispla in (318
m/z) and [M + H]+ o oxalipla in (398 m/z) and ca bopla in (372 m/z) (Figu e S3). O e all,
he abundance o he p ecu so ion o cispla in is lowe han o ca bopla in and oxali-
pla in p ecu so ions. Figu e 2 shows he SIM ch oma og am o a s anda d mix u e o he
h ee pla inized d ugs unde he op imal condi ions.
Figu e 2. LC/MS ch oma og am o a s anda d mix u e con aining 10 µg mL−1 o each o he pla inum
d ugs s udied.
2.2.2. MRM Mode
Ch oma og aphic condi ions we e he same as desc ibed o SIM mode. The MS de-
ec o was ope a ed in MRM mode, whe e p oduc ions a e agmen a ion a e used o
quan i a i e pu poses. Op imized LC-MS/MS pa ame e s o he de e mina ion o pla i-
na ed compounds a e gi en in Table 1. Adequa e agmen a ion is achie ed o ca -
bopla in and oxalipla in. Howe e , cispla in p esen s a low agmen a ion in he collision
cell. This issue, added o he ma ix e ec when applying he me hod o eal samples,
esul s in a small signal in he ch oma og am. To educe he ma ix e ec and imp o e
0
500,000
1,000,000
1,500,000
2,000,000
01234
Coun s
Adquisi ion ime (min)
Oxalipla in
0
250,000
500,000
750,000
01234
Coun s
Adquisi ion ime (min)
Ca bopla in
0
25,000
50,000
75,000
100,000
01234
Coun s
Adquisi ion ime (min)
Cispla in
Figu e 2.
LC/MS ch oma og am o a s anda d mix u e con aining 10
µ
g mL
−1
o each o he pla inum
d ugs s udied.
2.2.2. MRM Mode
Ch oma og aphic condi ions we e he same as desc ibed o SIM mode. The MS de-
ec o was ope a ed in MRM mode, whe e p oduc ions a e agmen a ion a e used o
quan i a i e pu poses. Op imized LC-MS/MS pa ame e s o he de e mina ion o pla i-
na ed compounds a e gi en in Table 1. Adequa e agmen a ion is achie ed o ca bopla in
and oxalipla in. Howe e , cispla in p esen s a low agmen a ion in he collision cell. This
issue, added o he ma ix e ec when applying he me hod o eal samples, esul s in
a small signal in he ch oma og am. To educe he ma ix e ec and imp o e he signal
Molecules 2022,27, 8139 5 o 11
in ensi y, di e en dilu ions we e applied o he ma ix, selec ing 1:10 as he mos op imal.
Figu e 3shows he MRM ch oma og ams.
Table 1.
Op imized LC-MS/MS pa ame e s used in he de e mina ion o cispla in, ca bopla in,
and oxalipla in.
Compound P ecu so Ion
(m/z)
P oduc Ions
(Quan i ie /Quali ie )
(m/z)
CE
(eV)
F agmen o
(V)
Cispla in 317.9 264.5/300.7 15/15 166
Ca bopla in 372.0 355.0/294.0 10/20 166
Oxalipla in 398.0 308.0/96.0 20/25 166
Molecules 2022, 27, x FOR PEER REVIEW 5 o 11
he signal in ensi y, di e en dilu ions we e applied o he ma ix, selec ing 1:10 as he
mos op imal. Figu e 3 shows he MRM ch oma og ams.
Table 1. Op imized LC-MS/MS pa ame e s used in he de e mina ion o cispla in, ca bopla in, and
oxalipla in.
Compound P ecu so Ion
(m/z)
P oduc Ions
(Quan i ie /Quali ie )
(m/z)
CE
(eV)
F agmen o
(V)
Cispla in 317.9 264.5/300.7 15/15 166
Ca bopla in 372.0 355.0/294.0 10/20 166
Oxalipla in 398.0 308.0/96.0 20/25 166
Figu e 3. LC/MS-MS ch oma og am o a s anda d con aining 10 µg mL−1 o cispla in, ca bopla in,
and oxalipla in.
This echnique, like he p e ious ones, allows sepa a ion in less han 4 min, as well
as simul aneous de e mina ion o he h ee compounds.
2.2.3. De i a iza ion and MRM Mode
To imp o e sensi i i y, de i a iza ion wi h DDTC was p oposed ollowing a sligh ly
modi ied p ocedu e as p e iously desc ibed [23]. Fo sample de i a iza ion, a 0.1 N
NaOH solu ion was p epa ed con aining 5% DDTC. Pla in de i a i es we e p epa ed by
adding 100 µL o a 5% DDTC solu ion o 500 µL o u ine. Samples we e homogenized by
0
500,000
1 ,00 0, 000
1 ,50 0, 000
2 ,00 0, 000
01234
Coun s
Adquisi ion ime (min)
Oxalipla in
0
50,000
100,000
150,000
200,000
250,000
01234
Coun s
Adquisi ion ime (min)
Ca bopla in
0
1000
2000
3000
4000
5000
6000
01234
Coun s
Adquisi ion ime (min)
Cispla in
Figu e 3.
LC/MS-MS ch oma og am o a s anda d con aining 10
µ
g mL
−1
o cispla in, ca bopla in,
and oxalipla in.
This echnique, like he p e ious ones, allows sepa a ion in less han 4 min, as well as
simul aneous de e mina ion o he h ee compounds.
2.2.3. De i a iza ion and MRM Mode
To imp o e sensi i i y, de i a iza ion wi h DDTC was p oposed ollowing a sligh ly
modi ied p ocedu e as p e iously desc ibed [
23
]. Fo sample de i a iza ion, a
0.1 N NaOH
solu ion was p epa ed con aining 5% DDTC. Pla in de i a i es we e p epa ed by adding
100
µ
L o a 5% DDTC solu ion o 500
µ
L o u ine. Samples we e homogenized by o ex o
15–20 s
andincuba ed o 15mina 45
◦
C[
23
]. In heseincuba ioncondi ions, hesul u o DDTC
eac swi hhyd a ed pla inum o o m heP -DDTC complex(
Figu e S2 Supplemen a y Ma e ial
).

Molecules 2022,27, 8139 6 o 11
A e incuba ion, 1400
µ
L o ace oni ile a e added o he sample o p ecipi a e he p o ein.
The sample was o exed o abou 15–20 s, and hen placed in o a mic ocen i uge o 15 s
a 14,500 pm. An aliquo o he uppe laye was injec ed in o he LC-MS/MS, p e ious
il a ion h ough a 0.22 µm nylon il e .
De e mina ion o de i a ized compounds was ca ied ou by LC-MS/MS. The mobile
phase included A: ace oni ile ( o mic acid 0.1% / ) and B: 10 mM ammonium o ma e
( o mic acid al 0.1% / ). Sepa a ion was pe o med unde he ollowing g adien elu ion
p og am: 0–0.5 min 5% A, 0.5–1 min 5–75 %A, 1–1.5 min 75–90% B, 1.5–2.5 min 90–95% A,
2.5–4 min 95% A, 4–5 min 95–100% A, 5–6 min 100% A, 6–7 min 100–5% A,
7–11 min 5% A
.
The injec ion olume was 10
µ
L and he column was se a a empe a u e o 35
◦
C. The
low a e was main ained a 0.4 mL min
−1
. The MS de ec o was ope a ed in MRM mode.
Op imized MS/MS pa ame e s a e shown in Table 2.
Table 2. Op imized MS/MS pa ame e s used in he de e mina ion o pla in de i a i es.
Compound P ecu so Ion
(m/z)
P oduc ions
(Quan i ie /Quali ie )
(m/z)
CE
(eV)
F agmen o
(V)
RT
(min)
P -DDTC 492 116.0/88.0 25/25 166 5.346
Two p oduc ions, one o quan i ica ion and o he one o con i ma ion, we e mon-
i o ed. Howe e , his me hod has no ob ious bene i in e ms o selec i i y, since he
h ee pla inized compounds o med he same adduc , binding only and exclusi ely o he
pla inum a om. The e o e, and al hough he sensi i i y o he echnique was excellen ,
being able o each a ew
µ
g mL
−1
, i was impossible o di e en ia e be ween he h ee
pla inized compounds and only he peak sum o he h ee adduc s was obse ed (Figu e 4).
Molecules 2022, 27, x FOR PEER REVIEW 6 o 11
o ex o 15–20 s and incuba ed o 15 min a 45 °C [23]. In hese incuba ion condi ions,
he sul u o DDTC eac s wi h hyd a ed pla inum o o m he P -DDTC complex (Figu e
S2 Supplemen a y Ma e ial). A e incuba ion, 1400 µL o ace oni ile a e added o he
sample o p ecipi a e he p o ein. The sample was o exed o abou 15–20 s, and hen
placed in o a mic ocen i uge o 15 s a 14,500 pm. An aliquo o he uppe laye was
injec ed in o he LC-MS/MS, p e ious il a ion h ough a 0.22 µm nylon il e .
De e mina ion o de i a ized compounds was ca ied ou by LC-MS/MS. The mobile
phase included A: ace oni ile ( o mic acid 0.1% / ) and B: 10 mM ammonium o ma e
( o mic acid al 0.1% / ). Sepa a ion was pe o med unde he ollowing g adien elu ion
p og am: 0–0.5 min 5% A, 0.5–1 min 5–75 %A, 1–1.5 min 75–90% B, 1.5–2.5 min 90–95% A,
2.5–4 min 95% A, 4–5 min 95–100% A, 5–6 min 100% A, 6–7 min 100–5% A, 7–11 min 5%
A. The injec ion olume was 10 µL and he column was se a a empe a u e o 35 °C. The
low a e was main ained a 0.4 mL min−1. The MS de ec o was ope a ed in MRM mode.
Op imized MS/MS pa ame e s a e shown in Table 2.
Table 2. Op imized MS/MS pa ame e s used in he de e mina ion o pla in de i a i es.
Compound P ecu so Ion
(m/z)
P oduc ions
(Quan i ie /Quali-
ie )
(m/z)
CE
(eV)
F agmen o
(V)
RT
(min)
P -DDTC 492 116.0/88.0 25/25 166 5.346
Two p oduc ions, one o quan i ica ion and o he one o con i ma ion, we e mon-
i o ed. Howe e , his me hod has no ob ious bene i in e ms o selec i i y, since he h ee
pla inized compounds o med he same adduc , binding only and exclusi ely o he pla -
inum a om. The e o e, and al hough he sensi i i y o he echnique was excellen , being
able o each a ew µg mL−1, i was impossible o di e en ia e be ween he h ee pla inized
compounds and only he peak sum o he h ee adduc s was obse ed (Figu e 4).
Figu e 4. LC/MS-MS ch oma og am o a s anda d mix u e con aining 0.1 µg mL−1 o each o he
pla inized compounds a e de i a iza ion.
3. Discussion
The ch oma og aphic condi ions used in each me hod we e condi ioned by he de-
ec o employed. Wi h UV-DAD, a good ch oma og aphic sepa a ion is key o iden i y he
analy es besides he UV spec um. The h ee pla inized cy os a ic d ugs we e pe ec ly
sepa a ed in e e sed phase using micella liquid ch oma og aphy, wi h 0.5 mM SDS as
0
50,000
100,000
150,000
200,000
0123456789
Coun s
Adquisi ion ime (min)
P -DDTC
Figu e 4.
LC/MS-MS ch oma og am o a s anda d mix u e con aining 0.1
µ
g mL
−1
o each o he
pla inized compounds a e de i a iza ion.
3. Discussion
The ch oma og aphic condi ions used in each me hod we e condi ioned by he de ec o
employed. Wi h UV-DAD, a good ch oma og aphic sepa a ion is key o iden i y he
analy es besides he UV spec um. The h ee pla inized cy os a ic d ugs we e pe ec ly
sepa a ed in e e sed phase using micella liquid ch oma og aphy, wi h 0.5 mM SDS as
he aqueous phase and MeOH as he o ganic phase in isoc a ic mode. In he MS/MS
de ec o , howe e , compound iden i ica ion was achie ed, besides i s e en ion ime, using
he a io be ween wo mass ansi ions (quali ie and quan i ie ). In mos o cases, he
Molecules 2022,27, 8139 7 o 11
c i e ia used a e hose p oposed by Commission Decision 2002/657/EC [
26
] which in ol es
ma ching
±
2% o e en ion imes and 80% ag eemen in ela i e ion a ios. Due o he
highe selec i i y o MS/MS de ec o , he sepa a ion o analy es is no so c i ical as occu s in
UV-DAD. In his case, ins ead o a su ac an as mobile phase, he use o an isoc a ic elu ion
wi h an aqueous bu e solu ion con aining 20 mM ammonium o ma e (pH adjus ed o 6.4
using o mic acid) (95%) (A) and AcN (5%) as he mobile phase was selec ed o imp o e
he ioniza ion o he p ecu so ions o he a ge compounds and hus sensi i i y.
On he o he hand, he use o in e nal s anda ds is a common p ac ice used o com-
pensa e he signal enhancemen o supp ession known o occu in elec osp ay ioniza ion
(ESI). I could no be used in his s udy since he pe cen age o ma ix e ec s obse ed was
no he same o all he compounds. As such, a ma ix-ma ched calib a ion cu e me hod
was used o o e come he ma ix e ec . In he case o he DAD me hod, because o he
high olume o sample injec ed, he use o an in e nal s anda d was no necessa y. Since
all p oposed echniques in ol e he di ec injec ion o u ine samples, he in luence o he
ma ix e ec was e alua ed by compa ison o calib a ion cu e slopes in pu e sol en (ex-
e nal calib a ion cu es) and calib a ion cu e slopes in u ine (ma ix-ma ched calib a ion
cu es). Calib a ion cu es we e p epa ed in iplica e a i e concen a ion le els in a
ange om me hod quan i ica ion limi s (MQLs) o 100
µ
g mL
−1
and we e cons uc ed
using analy e a ea (axis y) e sus analy e concen a ion (axis x). S uden ’s es was ap-
plied o e alua e s a is ical di e ences be ween ex e nal calib a ion and ma ix-ma ched
cu es. The calcula ed was compa ed wi h he wo- ailed abula ed alue,
ab
o he
app op ia e numbe o deg ees o eedom a P% con idence. Typical alues a e k = 2 o
95% con idence [
15
], so, i < k, he a io o he slopes is no signi ican ly di e en om 1,
and, i > k, he a io o he slopes is signi ican ly di e en om 1 and he ma ix calib a ion
mus be used. The calcula ed S uden ’s (2.93–14.8) showed s a is ical di e ences among
he slope alues o he calib a ion cu es and, consequen ly, he use o ma ix calib a ion
was necessa y.
The analy ical ea u es o he p oposed and op imized me hods we e calcula ed and
compa ed (Table 3). Linea i y was e alua ed in e ms o R
2
, p epa ing ma ix calib a ion
cu es in iplica e in he ange om he MQL o 100
µ
g mL
−1
. P ecision was calcula ed
as ela i e s anda d de ia ion (RSD%) o spiked samples in iplica e and selec i i y was
e alua ed as he abili y o p oduce esul s ha depend only on he analy e o be iden i ied o
quan i ied, wi hou in e e ence om o he species p esen in he sample. MDL and MQL
we e de e mined as he concen a ion, wi h a signal- o-noise a io o 3 and 10, espec i ely.
Table 3.
Quali y pa ame e s o he e alua ed me hods o de e mina ion o pla ina ed d ugs in
u ine samples.
Quali y
Pa ame e LC-UV(DAD) LC-MS
(SIM)
De i a iza ion
+ LC-MS/MS
(MRM)
LC-MS/MS
(MRM)
Linea i y
(R2)
Cispla in 0.999 0.991
0.998
0.991
Oxalipla in 0.999 0.996 0.992
Ca bopla in 0.998 0.991 0.994
P ecision
(RSD%)
Cispla in 1.5 12
8
7.9
Oxalipla in 2.0 10 7.6
Ca bopla in 5.1 15 12.8
Selec i i y No
(In e e ences) Yes No
(
Single p oduc
as sum) Yes
MDL
(µg mL−1)
Cispla in 1.5 1.5
0.0003
0.30
Oxalipla in 1.5 0.75 0.015
Ca bopla in 7.5 2.25 0.15
MQL
(µg mL−1)
Cispla in 5.0 5.0
0.001
1.0
Oxalipla in 5.0 2.5 0.05
Ca bopla in 25 7.5 0.50
Molecules 2022,27, 8139 8 o 11
In e ms o linea i y, co ela ion coe icien s we e highe han 0.992 o all he me hods.
P ecision, exp essed as RSD%, was less han 15% o all me hods.
Selec i i y is adequa e only o me hods employing LC coupled o MS. Wi h he
UV-(DAD) de ec o , he d awback ound was wo old: on he one hand, he pla inized
compounds do no p esen a cha ac e is ic abso p ion spec um ha would allow an un-
equi ocal iden i ica ion, and, on he o he hand, a high loss o selec i i y caused by he
p esence o in e e ences was obse ed when he me hod was applied o u ine samples.
U ine ma ix had o be highly dilu ed (1:50) o a oid loss o selec i i y and o be able o
quan i y he samples. In he me hod employing de i a iza ion, he DDTC binds exclusi ely
o a pla inum a om, which means ha he h ee analy es o m he same p ecu so . De i a-
ized cispla in, ca bopla in, and oxalipla in esul in a single adduc , so de e mina ion o
he indi idual analy es is no possible.
The lowes MQLs a e achie ed wi h he de i a iza ion me hod while he UV(DAD)
me hod has he highes one due o he la ge dilu ion o he sample ha was necessa y o e-
duce he ma ix e ec . In he MRM me hod, o ca bopla in and oxalipla in d ugs he e is a
g ea imp o emen in sensi i i y compa ed o he SIM mode ( om 7.5 o 0.50 and om 2.5 o
0.05
µ
g mL
−1
, espec i ely). The e o e, acco ding o he analy ical ea u es o he p oposed
echniques, he bes de e mina ion condi ions a e achie ed by LC-MS/MS MRM mode.
As he In oduc ion s a es, p e iously published wo ks a e ocus on he single de e mi-
na ion o a pla inized compounds, mainly cispla in, which may lead o lowe quan i ica ion
limi s (especially i a de i a iza ion s ep is being used, as can be seen in ou esul s in
Table 3). To he au ho s’ knowledge, he e is only one published wo k ha achie es his
aim. Recen ly, Dughe i e al. (2021) de eloped a me hod o he de e mina ion o cispla in,
ca bopla in, and oxalipla in in su aces. Howe e , besides being applied o a comple ely
di e en ype o sample, i employs zwi e ionic hyd ophilic in e ac ion liquid ch oma og-
aphy. This echnique p esen s some ad an ages, such as a highe sensi i i y han in
e e sed-phase LC when i is used wi h elec osp ay ioniza ion mass spec ome y. Despi e
his, i also p esen s some sho comings. Fo example, ma ix e ec in biological samples is
expec ed o be mo e p onounced in HILIC han in e e sed-phase LC due o he p esence
o phospholipids, which a e elu ed close o he peaks o in e es in HILIC. Fu he mo e, he
use o HILIC is no as ex ended as LC in e e se mode.
4. Ma e ials and Me hods
4.1. Chemicals and Reagen s
Analy ical quali y s anda ds (<99%) o ca bopla in, cispla in, and oxalipla in we e
pu chased om Sigma-Ald ich (S einheim, Ge many). LC-MS-g ade ace oni ile, MeOH,
and wa e we e supplied by Romil (Ba celona, Spain). Fo mic acid (98%) was p o ided by
Pan eac (Ba celona, Spain). Analy ical-g ade SDS, sodium die hyl ioca bama e ihyd a e,
and ammonium o ma e we e p o ided by Sigma-Ald ich (Mad id, Spain). NaOH was
pu chased om Pan eac (Mad id, Spain).
4.2. S anda d Solu ions
S ock s anda d solu ions o ca bopla in and oxalipla in we e p epa ed in MeOH:H
2
O
a 1000
µ
g mL
−1
and s anda d solu ion o cispla in was p epa ed in wa e 0.9% KCl (p/ ).
Each s anda d solu ion was kep a
−
18
◦
C in ambe glass bo les. Wo king solu ions
we e made by dilu ing o mixing indi idual s anda d solu ions o p oduce mix u es o
a ge compound.
4.3. Ins umen s
4.3.1. LC Coupled o UV-DAD De ec o
The LC-UV(DAD) sys em consis ed o a high-pe o mance liquid ch oma og aph
Agilen 1200 se ies (Agilen , San a Cla a, CA, USA) wi h high-p essu e bina y pump, au-
osample , and column o en coupled o a UV-DAD de ec o . Ch oma og aphic sepa a ion
Molecules 2022,27, 8139 9 o 11
was pe o med on a Zo bax Eclipse XDB C-18 Rapid Resolu ion (4.6
×
50 mm i.d., 1.8
µ
m)
column (Agilen , San a Cla a, CA, USA) he mos a ed a 30 ◦C.
4.3.2. LC Coupled o a MS De ec o
The LC-MS sys em was composed o a c wi h high-p essu e bina y pump, au osample ,
and he mos a ed column compa men , coupled o an Agilen 6495 se ies HPLC (Agilen ,
San a Cla a, CA, USA) coupled o a 6410 iple quad upole (QqQ) mass spec ome e wi h
elec osp ay ioniza ion.
Ch oma og aphic sepa a ion was ca ied ou ch oma og aphic sepa a ion using a
HALO C18 (50 mm
×
4.6 mm d.i., 2.7
µ
m) column he mos a ed a 30
◦
C and coupled o a
HALO C18 (5 mm ×4.6 mm d.i., 2.7 µm) gua d column.
MS de ec o was ope a ed using elec osp ay ioniza ion in he posi i e mode wi h he
sp ay ol age se a 4000 V. Ni ogen was used as nebulize gas, and nebulize p essu e
was se a 40 psi wi h a sou ce empe a u e o 250
◦
C. D ying gas (ni ogen) was hea ed o
350 ◦C and deli e ed a a low a e o 12 L min−1.
4.4. Sample Collec ion and T ea men
U ine samples we e collec ed om hospi al and belonged o oncology pa ien s ea ed
wi h di e en cy os a ic d ugs. Samples we e di ec ly injec ed, a e il a ion h ough a
0.22 µm nylon il e , excep o hose ha equi ed a p io de i a iza ion s ep.
5. Conclusions
Fou de e mina ion echniques, including LC sepa a ion coupled o UV-(DAD), MS in
SIM and MRM mode, and de i a iza ion wi h DDTC ollowed by MS/MS, we e op imized
o he analysis o he main pla inized cy os a ics (cispla in, ca bopla in, and oxalipla in) in
u ine samples. The LC-MS/MS me hod showed he bes esul s (R
2
< 0.991,
RSD% < 13
,
good selec i i y, and MDL < 1
µ
g mL
−1
). UV-(DAD) and de i a iza ion me hods ha e no
ob ious bene i in e ms o ma ix e ec and selec i i y, espec i ely, since in he i s case
he ma ix e ec p e en ed quan i ica ion in eal samples and, in he second case, he h ee
pla inized compounds o med he same adduc . Despi e he limi a ions o ha ing poo e
selec i i y, he de i a iza ion me hod o e s he ad an ages o i s high sensi i i y being able
o be used o an indi idual and speci ic analysis.
6. Applica ion
The LC-MS/MS me hod was applied o eal u ine samples belonging o pa ien s unde
ea men wi h pla inized d ugs. Resul s can be seen in Table 4. Cispla in, ca bopla in, and
oxalipla in we e de ec ed in he samples in concen a ions anging om 0.331 o
58.7 µg mL−1
.
Table 4.
Concen a ion o pla inum-based cy os a ic d ugs in u ine samples om oncologic pa ien s.
Sample Compound Concen a ion (µg mL−1)
01 Cispla in 11.6
02 Cispla in 7.34
03 Oxalipla in 0.33
04 Cispla in 8.43
05 Oxalipla in 11.9
06 Ca bopla in 42.5
07 Oxalipla in 1.07
08 Ca bopla in 58.7