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8/9/2019 Improved Sample Treatment (11)
http://slidepdf.com/reader/full/improved-sample-treatment-11 1/17
Improved
sample
treatment
for
the
determination
of
17
strong
sorbed
quinolone antibiotics from compost by ultra high performance liquid
chromatography tandem mass spectrometry
art ic l e
i nf o
Article history:
Received 14 December 214
Received in revised form
! "arch 21#
Accepted $ "arch 21#
Available online 12 "arch 21#
%ey&ords:
Antibiotics
'uinolones
(ompost
"icro&ave)assisted e*traction
"atri* e+ect
,-./(0""
13 Introduction
a b s t r a c t
he use of compost from se&age
sludge
for
agricultural
application is
no&adays
incre
sing5
since
composting is recogni6ed as one of the most important recycling options for this material5 bein
a source
of nutrients for plants but also of contamination by persistent pollutants3 In the present &or
5 a multi)
residue analytical method for the determination of 17 quinolone antibiotic residues in com
ost using
multivariate optimi6ation strategies and ultra high performance liquid chromatography0
tandem mass
spectrometry has been developed3 It is based on the use of micro&ave)assisted e*tractioat drastic
conditions
&ith
A(8:m)phosphoric
acid
91
&v;
for
#
mi n
at
12
<(5
in
order
to
achieve
a
quantitative
e*traction of the compounds 947$ of e*traction recovery;3 =*tracts &ere cleaned)up by
lt)assisted
liquid0liquid e*traction 9A//=; &ith 8a(l at p- 13# 9&ith -(l>4; and then using a dispers
e sorbent9.A;3 After /( separation5 the " conditions5
in
positive electrospray ioni6ation mode =I;5 &ere
individually optimi6ed for each analyte to obtain ma*imum sensitivity in the selected rea
tion mon)
itoring mode 9R";3 he analytes &ere separated in less than 7 min3 (incophen &a s used
s surrogate
standard3
he
limits
of
detection
ranged
from
32
to
3#
ng
g?1
5
and
the
limits
of
the
quanti@c
tion from
3# to 13# ng g?1
5 &hile intra) and inter)day variability 9 RD; &a s under 7 in all cases3 A
recovery
assay
&a s performed &ith spied samples3 Recoveries ranging from #3! to 1$32 &ere obtaind3 (leanup
procedure reduced signi@cantly matri* e+ects5 &hich constitutes an important achievement5 c
nsidering
the important dra&bacs of matri* components in quality and validation parameters3 hi s mhod
&a s
applied to several commercial compost samples3 >nly $ of the studied antibiotics &ere not
etected in
any of the samples3 he antibiotics &ith the highest concentrations &ere ciproBo*acin 9
!$ ng g?1
;5
oBo*acin 971
ng
g?1
;5 and enroBo*acin 9$74
ng
g?1
;5 &hich &ere also the only ones fou
in all theanaly6ed samples3 he results sho&ed that this method could also be potentially adapted for tanalysis
of other strong
sorbed basic pharmaceuticals in solid environmental matrices3
21#
=lsevier
E3F3
All
rights
reservd3
In recent years5
antibiotics
have been identi@ed as an em
erging
class
of
potential
contaminants3
As
these
compounds
arehighly
Antibiotics are &idely used in human and veterinary me
dicine
G1H 3
After
administration5
from
1
to
of
the
drug
is
e*cre
ted in
urine or feces into se&age G2H 3 ubsequently5 these substan
ces are
continually introduced into
the environment5 mainly thro
gh &as)
te&ater
ef Buent
from
municipal
treatment
plants5
hospita
or
live)
stoc activities G!0
#H3 hese compounds can also reach
terrestrial
8/9/2019 Improved Sample Treatment (11)
http://slidepdf.com/reader/full/improved-sample-treatment-11 2/17
environmental
compartments
&hen
sludge
is
spread
on
the
s
oil as
fertili6er3
n (orresponding author3 el3: I!4 #C 24 7 C J fa*: I!4 #C 24 !! 2C3
=)mail address: a6afraKugr3es 9A3 Lafra)MNme6;3
ttp:d*3doi3org1311$O3talanta321#3!311
!)14 21# =lsevier E3F3 All rights reserved3
bioactive5
concerns
have
arisen
about
the
presence
and
ossible
harmful
e+ects
of
these
substances
G1H 3
ome
scienti@c
studi
have
demonstrated adverse e+ects from longstanding5
lo&)dose e*po)
sures
in
both
aquatic
and
terrestrial
&ildlife3
here
is
also
a
s
rious
interest over
their
role
in
enhancing
antibiotic
resistance
among
pathogenic bacteria5 rendering current antibiotics ine+ective
n
the
treatment of numerous diseases G$H 3 Antibiotics can also a+
ct the
endocrine
system
of @sh
and
be
to*ic
to
algae
and
invertebra
es3
'uinolones are one of the maOor
classes of antimicrobialthat
are
employed
&orld&ide
in
human
and
veterinarian
me
icine3
According to a categori6ation of antibiotics by the Porld -
ealth
>rgani6ation in 2115 9Buoro;quinolones are included as
uman
24C 83 Dorival)MarcQa et al3 alanta 1!C 921#; 24702#7
critically important antibiotics3 he panish Agency of "edicinesand -ealth .roducts reported in 2 G7 H a continual use
of qui)
nolones
along
the
last
2
years3
(iproBo*acin
and
oBo*acin
a
re
the
most used quinolones
in
hospitals
GC H
and
livestoc
G H5 &hile
enroBo*acin is the most used drug in veterinary medicine
G$ 5H3
Administered quinolones are largely e*creted as unchanged
com)
pounds
in
urine5
and
consequently
discharged
into
se&age
G
151H3 hese
antibiotics
are
not
completely
removed
at
PP.s
and
their
continuous introduction into the environment maes them
per)
sistentS compounds G11H3 'uinolones that are initially pres
ent in
&ater bodies may
be transferred &ithout degradation and stored
in di+erent environmental compartments G12H3 "oreover5 hu
mans
may
be
e*posed
to
residues
of
drugs
in
the
environmen
t by a
number of routes including the consumption of crops that
haveaccumulated these substances from fertili6ed soils G1!H3
he utili6ation of se&age sludge for agricultural applicat
ion is
increasing G14H5 and composting has been recogni6ed as one
of the
best
se&age
sludge
recycling
options
G1#H3 .revious studie
s have
sho&n that the degradation of some pharmaceuticals and pe
rsonal
care
products
may
tae
place
during
biosolids
composting
G1$
H5
but
still
very
fe&
systematic
&ors
concerning
the
degradation
of
anti)
microbials during se&age sludge composting have been developed
G1701H3
Instead
of
developing
technologies
to
assist
the
degradation
of
antibiotics
in
soil
and
preventing
them
from
contaminating
&aers
and
crops5
it
is
more
e+ective
to
eliminate
the
antibiotics
in
com
ost
before it is applied to agricultural land as fertili6er G2H suc
that
composting
process
may
provide
a
practical
and
economical
solu
on
for
reducing
the
ris
of
pollution
in
the
environment3
Although several papers on the determination of antibioti
inse&age
sludge
have
been
reported
in
the
scienti@c
literature
G21
24H5
only
fe&
have
been
published
about
their
determination
in
comostor treated sludge G2# 02CH5 or furthermore the speci@c e*tract
n of
quinolones
&ith
good
recoveries5
considering
the
strong
interactns
that these compounds usually form
&ith matrices &ith a
large
charge of organic matter G2#H3 he reported methods include
di+)
erent sample treatment procedures5 such as micro&ave)
assisted
e*traction
9"A=;5
ultrasonic
e*traction
9,=;5
pressuri6ed quid
e*traction 9./=;5 matri* solid phase dispersion 9".D; or st
bar
sorptive e*traction 9E=;3 -o&ever5 most of these
&ors app
es a
further
cleanup
step
by
the
traditional
solid)phase
e*traction
9.=;5
&hich
is
a
tedious5
costly
and
time)consuming
procedure3
In
the
last
years5 modern sample preparation techniques5 such as 'uic5
=asy5
(heap5 =+ective5 Rugged and afe 9'u=(h=R;5 based on the
mini)
mi6ation of organic solvents and less
time consumption have een
8/9/2019 Improved Sample Treatment (11)
http://slidepdf.com/reader/full/improved-sample-treatment-11 3/17
developed
G2H3
his
technique
is
based
on
salt)assisted
liquid0
liquid
e*traction
9A//=;
and
it
has
demonstrated
to
be
a
good
alternative
to .= as a cleanup step3 After e*traction5 the organic solvent
phase
containing
the
analytes
of
interest
is
cleaned
up
again
by
addi
ng
an
.=
sorbent
9dispersive
.=5
D).=;
such
as
primary
secondary
amine
9.A;
or
(1C5
to
remove
interferences3
Recently5
some
&ors
f
or
the
determination
of antimicrobial
agents using this techni
que
in
environmental matrices have been published G!0!4H5 in
cluding
quinolones G!15!4H 3 -o&ever5 the e*traction procedure led to lo &
recoveries5
&hich is indicative of the need of drastic
e*
traction
conditions3
he aim of the present study &as to develop a multi)
residue
analytical
method for
the determination
of 17
quinolones5 &i
dely
used
in
human
and
veterinarian
medicine5
in
compost
samples
at
trace levels3 he method includes an improved sample trea
tment
that consists
in micro&ave)assisted
e*traction &ith high
recov)
eries
for
strong
sorbed
quinolones5
follo&ed
by a
cleanu
p
step
based on
A//= and D).= to reduce matri* e+ects and
conse)
quently to increase the analytical sensitivity3 he determi
nation
&a s
performed
by
,-./(0""3
he
method
&as
appli
ed
to
compost
samples
obtained
from
se&age
sludge3
h is
analytical
method
is
useful
for
the
development
of
more
in)depth
studies on
the
occurrence
and
fate
of
these
antibiotics
during
the
compostngprocess
and
their
potential
re)entry to
the
environment3
23 "aterials an d methods
2313
(hemicals
and
reagents
Pater 91C32
"T
cm; &as puri@ed using a "illi)' system f
om
"illipore 9Eedford5 "A5 ,A;3 Analytical grade standards of quno)lones: pipemidic acid 9.I.;5 eno*acin 9=8>;5 norBo*acin 9
>R;5
ciproBo*acin
9(I.;5
oBo*acin
9>U/;5
enroBo*acin
9=8R;5
diB*acin
9DIU;5
marboBo*acin
9"AR;5
danoBo*acin
9DA8;5
saraBo*acin
9
R;5
cino*acin5 9(I8;5 lomeBo*acin 9/>";5 mo*iBo*acin 9">V;5 nali
*ic
acid 98A/;5 o*olinic acid 9>V>;5 Bumequine5 9U/,;5 piromidic acid
9.IR;J the surrogate 2)phenyl)4)quinoline carbo*ylic acid 9ci
co)phen5 (I(;5 and the internal standard5 ca+eine 9(AU; &ere purch
sed
from igma)Aldrich 9t3 /ouis5 ">5 ,A;3 Individual standard s
lu)
tions
of
compounds
92 Wg
m/?1
;
&ere
prepared
in
a
&
ater
methanol mi*ture 91:45 vv; and stored at ?2
<(3 hese soluions
&ere prepared fresh monthly3 Poring standard mi*tures ere
prepared
by
diluting
the
individual
stoc
solution
in
methanol
o
n
the
initial
mobile
phase
immediately
before
use3
hese
solu
ons&ere stored at 4
<( and prepared fresh &eely3 All solutions
ere
stored in dar glass bottles to prevent photodegradation3 echcal
acetonitrile
9min3
pure;
used
for
e*tractions
&as
purchased
om
FPR
9Radnor5
.ennsylvania5
,A;3
m).hosphoric
acid
9!!3#0!$3#
as
-.>!;5
perchloric
acid
9$
&&;5
anhydrous
magnesium
sulfate
ndsodium chloride &ere provided by .anreac 9Darmstadt5 Mermny;3
.A sorbent 9primary secondary amine5 40$ mm; &as purch
sedfrom charlab 9Earcelona5 pain; and EA%=RE>8Ds octadecy(1C
sorbent 94
mm particle si6e; &as provided by X33 Eaer 9Deve
nter5
he 8etherlands;3 /(0" grade &ater and methanol5 aceton
rile5
sodium
hydro*ide5
ammonia
9L2#;
and
formic
acid
9LC;Yused
for
the
preparation
of
standards5
mobile
phases
and
p-
adOustmets
Y&ere
purchased
from
Ulua
9t3
/ouis5
">5
,A;3
2323 Instrumentation and soft&are
he
e*traction &as
performed
&ith
a "ilestone
9=->
=/;
8/9/2019 Improved Sample Treatment (11)
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micro&ave solvent e*traction /abstation 9heldon5 (5 ,A;5
oper)
ating at 24## "-6 &ith a ma*imum delivered po&er of 1
P3
he
time5
temperature
and
micro&ave
po&er
control
&ere
adOu
sted
and
controlled
throughout
the
process
using
a
control
terminal
that
runs =asy(>8R>/ soft&are3 emperature &as monitored &ith
the
aid
of
a
thermopar
A()4
temperature
sensor3
his
apparatus
&as
equipped
&ith
1
closed
vessels
made
of
teBon5
for
&hic
h5
the
ma*imum
operating
temperature
&as
!
<(3
,-./(0""
analysis
&as
performed
using
a
Paters
Acquity
,./(Z -)(lass
9Paters5
"anchester5
,%;5
consisting
of
an
A(',I[
,./(Z
binary
solvent
manager
and
an
A(',I[
,./
(Zsample
manager3
eparation
of
compounds
&as
obtained
&ith
A(',I[
,./(
E=-Z (1C
column
9137 Wm J
231
mm \
1
mm ;
9Paters5 ,%;3 A Vevo
'
tandem quadrupole mass spectromter9Paters; equipped &ith an orthogonal L)sprayZ electros
ray
ioni6ation 9=I; source &as used for antibiotic detection3
Uor p- measurements5 a =,=(- .(D $# digital p-)meter
&ith
a
combined
glass0AgAg(l
9%(l
!
";
electrode
9=,=(-
Instruments/td5
ingapore;
&as
used3
(ompost
samples
&ere
free6e)dried
using
a
(A8FA(
(oolafeZ
free6e
dryer
9/ynge5
Denmar;3
A vo
te*)
mi*er
9I%A5
taufen5
Mermany;5
a
Digicen
21
centrifuge
9>rto
Alsa5
"adrid5 pain;5 an ,ltrasons)-D ultrasound bath 9electa5
Earcelona5
pain;5 a pectrafugeZ 24D centrifuge from /abnet Internat
onal5
Inc3 98e& Xersey5 ,A; and a sample concentrator 9tuart5 taord)
shire5 ,% ; &ere also used3 tatgraphics .lus soft&are versio
#3183 Dorival)MarcQa et al3 alanta 1!C 921#; 24702#7 24
9tatpoint echnologies Inc35 Firginia5 ,A; &as used for statisticaltreatment
of
data3
23!3
Easic
procedure
After collection5 samples &ere free6e)dried and sieved to
r1341
mm
mesh
si6e
and
introduced
in
dar
glass
bottl
es
and
stored
in
the
dar
at
4
<(
until
analysis3
23!313
.reparation
of
spied
samples
Uor
spied
samples
preparation
during
optimi6ation
of
e*tra
ction
parameters5
1
m/
of
a
methanolic
standard
solution
of
analyte
s
&as
added
to
1
g
of
compost
to
obtain
a @nal
concentration
of
C
n
g
g?1
for
each
antibiotic3
his
volume
allo&s
the
analytes
to
come
in
contact &ith the &hole sample3 In order to attain sorption
equili)
brium
and
to
allo&
complete
evaporation
of
methanol
from
sa
mples5
the mi*tures &ere shaen on a vorte* mi*er for 1 min an
d &erethen left to stand for 24
h at room temperature in
the dar
before
analysis3
Uor
method
validation
9recovery
assays5
precision5
and
true
ness;5
blan samples &ere spied at di+erent concentrations by
adding
1
m/ of the spiing standard solution containing the analyt
es and
the
surrogate
9(I(; to
1
g of
compost
samples3 hen5 the
spied
samples
&ere
treated
as
it
&as
indicated
before3
he
blan
sa
mples
&ere
previously
analy6ed
in
order
to
ensure
the
absence
of
analytes
or that these &ere belo& the />Ds of the method3 -o&ever
none
of
the
analy6ed
compost
samples
from
se&age
sludge
&ere
fo
nd to be
completely free of analytes and therefore a di+erent typ
of com)
mercial compost &as used as blan3 A compost type that es
ablished
very similar interactions &ith the target analytes &a
selected5
&hich resulted in comparable e*traction recoveries a
d
matri*e+ects5
as
sho&n
in
Uig3
1
9supplementary
material;
or
a
con)
centration
of
1#
ng
g?1
for
each
antibiotic3
23!323
=*traction procedure
A
sample
of
13
g
of
compost
&a s
placed
in
a
micro&av
vessel5
1#
m/ of the solvent5 A(8:m)phosphoric acid 1 97:!5
v; &ere
added3 amples &ere e*tracted for #
mi n 91
mi n for h
lding; at
12 <( and 1
P of po&er3 en vessels &ere process
d simul)
taneously3
In
the
=thos
unit5
the
pressure
is
controed
auto)
matically5 and overpressure complications are thus avoi
ed3 >nly
one e*traction cycle &as required3 After micro&ave irradi
tion5
an
air Bo&
cooled
the
vessels
inside
the
micro&ave
unit
9o4
<(;3
In
order
to
decrease
the
matri*
co)e*tractives
in
the
eract
that
could cause the matri* e+ect5 a cleanup of the e*trac
based on
A//=
and D).=
&as
carried
out3
he
e*tract
&as
transferred
to
a
#
m/
Ualcon
conical
tube
containing
!
g
of
8a(l3 hen
7#
m/ of
8/9/2019 Improved Sample Treatment (11)
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perchloric
acid
$
&a s
added3
he
mi*ture
&as
hand)shaen
for
2 mi n and centrifuged for ! mi n at 2#4g3 he organic phase
9A(8;
containing
the
analytes
&a s transferred
to a
ne &
Ualco
n
tube
containing !
mg .A and $
mg anhydrous "g>43 he
mi*)ture
&as hand)shaen for 1
mi n and centrifuged
for
1
mi n
at
2#4g3 he supernatant &as decanted into a glass vial an
d5 afterevaporation
to
dryness
at
#
<(
under
a
82
stream5
the
resid
ue
&as
dissolved in #
m/ of a solution of ammonium formate 2
#
m"
9p-
!3;:methanol
91:15
vv;5
centrifuged
at
1$5!g
for
!
min and
inOected
into
the
/(
system3
2343
,ltra
high
performance
liquid
chromatography0tandem
mass
spectrometry
conditions
(hromatographic separation &as performed using a binary gra)
dient
mobile
phase
consisting
of
ammonium
formate
2#
m"
sol
ution
at p- !3 9solvent A; and methanol 9solvent E;3 he Bo& r
ate &as
! m/
min?15
the
column
&as
maintained
at
4
<(
and
the
inO
ection
volume
&as
7 m/3
Mradient
conditions
&ere
as
follo&s:
initial
mobile
phase5
C
9A;5
&hich
&as
linearly
decreased
to
!
9A&ithin
!3
min and to &ithin 31
min and held for 13
min to c
ean the
column
using
1
organic
mobile
phase3
Uinally5
bac
to
C
9A;
in
31
min
and
ept
for
13
min
to
equilibrate
the
column3
otal
ru
time
&as
7
min3
he
mass
spectrometer
9";
&as
operated
&ith
elect
spray
ioni6ation
9=I;
in
positive
ion
mode
and
in
multiple action
monitoring 9"R"; mode3 he "" parameters &ere optim
6ed
individually
for
each
antibiotic
by
infusion
of
3# Wg
m/?1
sta
dard
solution
in
the
initial
mobile
phase5
using
G"I-HI as
precurso
ion5
under
combined mode3 Instrument
parameters
&ere
as fllo&s:
capillary voltage5 3$
F J source temperature5 1#
<(J des
lvation
temperature5
#
<(J
cone
gas Bo&5
1#
/
h?1J
desolvation
g
s Bo&5
#
/
h?1J
collision
gas Bo&5
31#
m/
min?15
and
nebuli6er
g
s Bo&5
73
bar3 8itrogen 9L3#; &as used as a cone and des
vation
gas5 and argon 93; &as used as a collision gas3 After te
pre)
cursor
ions
&ere
selected5
product
ions
&ere
obtained
&ith
a
com)
bination of collision energies and cone voltages3 able 1 sho&
s the
t&o
most
sensitive
transitions
9one
used
for
quanti@cation
a
d
the
other
for
con@rmation;
selected3
o
obtain
the
ma*imum
sens
ivity5the
most
abundant
transition &as
used
for
quanti@cation
D&ell
time for each transition &as 2# ms5 and interscan delay &aset at
!
ms3 Data acquisition &as performed under time)segmente
con)
ditions5
based
on
the
chromatographic
separation
of
compoun
s5 to
ma*imi6e the sensitivity3 he optimum collision energie
cone
voltages for each transition5 segment periods and retention tim
s are
also summari6ed in able 13
!3
Results
an d
discussion
!313
>ptimi6ation of the
micro&ave)assisted
e*traction
he optimi6ation of the e*traction method &as based on apre)
vious
&or
about
the
determination
of
quinolones
in
se&age
s
udge
G2!H5
&hich
&as
improved
by
applying
a
cleanup
process
to
th
"A=
e*tracts
based
on
'u=(h=R
technique5
since
these
e*tracts
ontain
high amounts of matri* components due to the drastic conitions
8/9/2019 Improved Sample Treatment (11)
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that
are
applied
during
the
e*traction
step3
ErieBy5
samples
of
1
g
of
compost
previously
spied
&ith
the
analytes
&ere
e*tracted
for
17
min
at
C7
<(
and
1
P
using
A(8:aqueous
bu+er
"cIlvaine
p-
!
91:15
vv;
as
e*traction
solvent3
hen5
cleanup
&as
performed
using
a mi*ture of 2
g of "g>4 anhydrous and 1
g 8a(l3 he org
anic
phase
containing
the
analytes
&as
separated
and
dried
&ith
$ mg
"g>43
he
e*tract
&as
evaporated
to
dryness
at
#
<(
under
a
82
streamJ
dissolved
in
# m/
of
ammonium
formate
2#
m"
9p-
!3
;:
methanol5
1:15
vv5
containing ca+eine as
internal
standard
9#
ng
m/?1
;J centrifuged at 1$5!g for !
min and inOected
into
the /( system3 (a+eine &as used as internal standard during
the
optimi6ation process5 because it &as the most appropriated co
m)
pound
to
control
variations
during
chromatographic
analysis5
s
ce
it
produces
a
very
sensitive
and
constant
signal5
besides
that
it
&
as
not
naturally found in compost samples that &ere used for th
opti)
mi6ation
process3
It &as observed that using this basic procedure5 ve
y lo&
recoveries &ere
obtained for
the
antibiotics
in
compost s
mples9o2;3
herefore5
it
&as
decided
to
replace
the
bu+er
"cIlva
e
by
an
aqueous
solution
of
m)phosphoric
acid
3!
9&v;
that
hasbeen
already applied for the determination of quinolones in
everal
matrices
G!#H3
he
use
of
a
mi*ture
of
A(8:m)phosphoric
aci
3!
9&v;
as
e*traction
solvent
produced
adequate
results
and
reco
eries
increased around 73 A(8 &as ept as organic solvent an
other
e*traction parameters &ere optimi6ed using chemometric
chni)ques to improve antibiotic recoveries3
2# 83 Dorival)MarcQa et al3 alanta 1!C 921#; 24702#7
able 1>ptimi6ed R" conditions and retention times for quinolones5 internal standard and surrogate3
(ompound R"1a
(F(=b
R"2a
(F(=b
Ion ratio egment 9min; R 9D;c 9min;
(AU 9I; 1#31)1!73 !21C 1#31)13 !222 37 23043 !32 93#;
(I( 9M; 2#32)1273 4!4 2#32)2223 42$ 3$2 43073 #3#1 93!;
Amphoteric quinolones
.I. !432)21731 122 !432)2C$32 11C 341 23043# !317 934;
"AR !$!32)713 12 !$!32)!231 114 3! 23043# !3!! 93!;
=8> !2132)
!!32 $1C !2132)
2!23 $!4 3!1 23043# !3$1
93$;>U/ !$232)!1C32 41C !$232)2$131 42$ 37 23043# !3$2 93!;
8>R !232)!232 !C1C !232)27$32 !C1$ 31C 23#0#3# !3C! 93#;
(I. !!232)!1432 141C !!232)2!131 14!4 31 23#0#3# !3C 934;
DA8 !#C32)!432 224 !#C32)C13 2!$ 34! 23#0#3# !34 934;
/>" !#232)2$#31 $22 !#232)!C32 $1$ 3$2 23#0#3# 432 93!;
=8R !$32)!1$32 C1C !$32)24#31 C2$ 3## 23#0#3# 43# 934;
AR !C$34)!$C32 #2 !C$34)!4232 #1C 32! 23#0#3# 432C 93#;
DIU 432)!C232 222 432)!#$32 21C 3C# 23#0#3# 43!# 93!;
">V 4232)!C432 !22 4232)!#C32 !1C 3$ 23#0#3# 4374 932;
Acidic quinolones
(I8 2$!32)24#31 414 2$!32)1C3 42$ 3#C 23#0#3# 43#! 93!;
>V> 2$232)2443 41C 2$232)21# 42$ 322 43073 #34 93!;
8A/ 2!!31)21#3 214 2!!31)1C73 224 3!$ 43073 #3#7 93#;
U/, 2$232)2443 $1C 2$232)223 $!2 3C 43073 #3$$ 932;
.IR 2C32)
27131 C1C 2C32)
24!31 C2C 372 43073 #3C#
931;
I: internal standardJ M: surrogate3
aR"15 transition used for quanti@cationJ R"25 transition used for con@rmation3
b (F5 cone voltage 9F;J (=5 collision energy 9eF;3
c R5 Retention timeJ D5 tandard deviation3
!31313
creening
by
t&o)level
half)fraction
design3
Uirst5
in
order
to
select
the
most
inBuential
variables5
a
t&o
level
half fractional factorial e*perimental design 92#?1
; &as perf ormed5resulting in 1 e*periments 9i3e3 1$I! central points;3
olvent
composition5
p-
of
e*traction
solvent5
e*traction
solvent
volume5
temperature
and
e*traction
time
&ere
varied
at
di+erent
evels
9Uig3
1A;3
he
studied
response
&as
the
e*traction
recovery3
ab
e
1
9supplementary material; sho&s the used screening design ma
ri*3
.areto
charts
&ere
obtained
and
statistically
signi@cant
e+ec
s
of
the variables &ere screened
using a
tudent]s
t)test for A>FA3Fariables having a con
@dence greater than
#3 &ere cons
ered
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to
have
a
signi@cant
e+ect
on
the
e*traction
ef @ciency3
Uig3
1E
and
(
sho&s
the
statistically
signi@cant
e+ect
of
each
variable5
pre
sented
as
the
sum
of
recoveries
of
quinolones
that
&ere
grouped
into
t&o
classes:
amphoteric
and
acidic
quinolones3
he
evaluated
variables
and
some
of
their
interactions
res
ulted
signi@cant3 emper atur e and solvent composition &ere the
most
inBuential
parameters5
follo&ed
by
p-
of
the
solvent5
&hereas
e*traction
time
&as
more
signi@cant
for
acidic
quin
olones3
Although
the
volume
of
the
e*traction
solvent
&as
also
signi
@cant
in
both
cases5
its
interactions
&ere
not
important3
herefore5
it &a sdecided
to @*
this
variable
to simplify
the
optimi6ation
of
sig)
ni@cant factors3 Folume &a s set at 1#
m/5 corresponding to the
central
point
value3
he
most
important
interactions
are
associated
to temperature5
solvent composition and
e*traction
time5 being
the
most
signi@cant
the
positive
interaction
bet&een
temper
ature
and solvent composition3 In order to mae practical the e*traction
procedure5
the
number
of
e*traction
cycles
&a s
set
at
13
!31323
>ptimi6ation
of
signi@cant
parameters
by
a
Doehlert
d
esign
he most inBuential variables 9solvent composition5 p- of the
e*traction solvent5 temperature and
e*traction time; &er
e
opti)mi6ed using a Doehlert e*perimental design3 he Doehlert
matri*
consisted of 2! e*periments5 including three central points3
even
levels
for
temperature
9from
#
to
12 <(;
and
solvent
compo
sition
94
to
1
A(8;5 @ve
levels
for
m)phosphoric
acid
concentration
9from
3!
to
1
&v;
and
three
levels
for
e*traction
time
9from#
to
2
min; &ere considered3 A &ide temperature range &as ap
ied5
considering the chemical stability of this family of antibiotics
!$H 3
he
e*traction
conditions
for
each
of
the
2!
e*perimental
runs
inthe
Doehlert
matri*
are
sho&n
in
able
2
9supplementary
materia
3
he data &ere evaluated by A8>FA and the test gave d
er)
mination
coef @cients
9R2
;
bet&een
3C$!
and
3C3
inc
the
.
values
for
the
lac)of)@t
test
&ere 43#
in
all
cases5
the
model
appears
to
be
satisfactory
&ith
the
#
of
con@dence
level3
'uadratic
terms
for
temperature
and
e*traction
time
restedstatistically signi@cant for all compounds3 Uor solvent compos
ion5quadratic
terms
&ere
signi@cant
only
for
amphoteric
quino
nes3
olvent
composition
for
acidic
quinolones
and
m)phosphoric
cidconcentration
&ere
signi@cant
only
in
their
lineal
terms3
he
obse
ved
tendency
for
both
groups
of
analytes
&as
that
the
solvent
compsi)
tion for acidic quinolones5 temperature5 and e*traction time h
d a
positive e+ect5 &hereas solvent composition for amphoteric q
no)
lones
had
a
negative
e+ect3
Important
interactions
&ere
also
obse
ved
bet&een
solvent
composition
and
temperature
9positive;5
so
ent
composition and m)phosphoric acid concentration 9negative;3
!313!3
>ptimi6ation of
multiple
responses
he optimi6ed conditions obtained using this approach usu
lly
conBicted &ith the aim of achieving good yields of analysis r
cov)
eries for all the studied compounds5 especially &hen some d+er)
ences bet&een both the groups of quinolones &ere found3 h
s5 a
multicriteria)decision maing process is essential and a total d
sir)ability function5 D5 &as used to optimi6e all responses simultaneo
sly
9the recoveries of the 17 analytes;3 his function is a meas
re of
overall
quality
and
provides
conveniently
a
&ay
to
compare
sev
ral
responses
and
to
select
the
optimum
&ith
the
most
desirable
pr
p)
erties3 Desirability ranges from 6ero to one3 ,nder the ment
ned
optimi6ation criteria5 the e*perimental conditions correspondin
to
one ma*imum in the desirability function 9D^321; &ere so
ent
83 Dorival)MarcQa et al3 alanta 1!C 921#; 24702#7 2#1
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Uig3
13
9A;
/evels
for
the
factors
e*amined
in
the
2#?1
screening
design3
tandardi6ed
main
e+ect
.areto
charts
for
9E;
amphoteric
quinolones
and
9(;
ac
c quinolones3 9I;
.ositive e+ects on the response5 9?; negative e+ects on the response3 Fertical line sho&s the limit of decision to consider the signi@cance of the f
ctors 9based on the
standardi6ed e+ect^estimated e+ectstandard error5 at # of con@dence level;3
composition5 7 A(8J m)phosphoric acid concentration5 1 &vJtemperature5 12
<(J
e*traction
solvent
volume5 1#
m/ J
e*
traction
time5
#
min
and
1
cycle
of
e*traction3
he
response
surfaces
obtained
for
the
global
desirability
fun
ction
are
presented
in
Uig3
23
hese
plots
&ere
obtained
for
a
given
pair of
factors5
&hile
maintaining
the
other @*ed
at
their
optimal
valu
es3
he
desirability
is
6ero
&hen
solvent
composition
is
higher
than#
A(8
9Uig3
2A;5
and
bet&een
4
and
#
A(83
his
con@r
ms
the
importance
of
establishing
an
adequate
ratio
organicaqueous
phases
for
the
e*traction
solvent5
considering
the
recogni6ed
&ater
sol
ubility
of quinolones
at
the
p-
conditions
that
are
e*tracted3
>n
the
other
hand5 higher desirability
is
observed at
higher
concentrati
ons
of
m)phosphoric acid5 &hich is an indication that strong acidic
condi)
tions are required for the ef @cient
e*traction of the targe
analytes3
emperature is an important factor 9Uig3 2E;5 since as temperatureincreasesJ
the
desirability
also
becomes
signi@cantly
higher
probably
because
higher
temperature
can
brea
ef @ciently
the
ecogni6ed
strong
interactions
bet&een
quinolones
and
the
matri*3
!323
>ptimi6ation
of the
cleanup procedures
by
A//=
and
D).=
sorbents
ince a
drastic
e*traction
technique
as
"A=
&a s
used5 theresulted
e*tract
usually
contains
a
signi@cant
proportion
matri*
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components5
and
it
requires
cleanup
prior
to chromatographicanalysis in order to reduce matri* e+ects
and increase met
hod
sensitivity3 It &a s
decided to apply
a
cleanup procedure
based
on
'u=(h=R5 &hich consists in t&o di+erent
steps:
the @rst
one
by
A//=5
follo&ed
by
the
use
of
a
dispersive
.=
sorbent3
!32313
>ptimi6ation
of
cleanup
by
A//=
A//=
is
a
procedure
that
involves
&ater)miscible
solvents
and
is
based
on
the
salting)out
e+ect3
he
term
also
connotes
reduction
of
mutual
miscibility
of
t&o
liquids
by
the
addition
of
electroly
tes3
Although A(8 is miscible &ith &ater in any proportion at ro
om
temperature5 the addition of salt signi@cantly reduced the mu
tual
miscibility5
even
resulting
in
phase
separation
of
A(8
from
aqueous
phase3 he
separation is from
homogeneous
solution inst
ad
of
through
emulsion
or
suspension
and
centrifugation
can
facilita
e
the
process3 hus 5 matri* components5 mainly &ater)soluble5 co
ld be
separated
from
analytes
that
migrate
to
organic
layer3
Uig3
!A
ho&s
the
results
of
the
e*periments
performed
to
determine
the
e+
ct of
the type
of added salt on the partitioning of the antibiotics
n the
upper
layer3
hree
salts
&ere
evaluated:
8a(l5
"g>4
and
98-
2>45as
&ell
as
combinations
of
them5
as
it
happens
&hen
methodsbased
on
'u=(h=R
are
applied
98a(l0"g>4;3
8a(l
&as
selected
as
the
optimum
salt
for
A//=3
"g>4
ad anegative e+ect5 even though this is a common)used saltg)out
2#2 83 Dorival)MarcQa et al3 alanta 1!C 921#; 24702#7
Uig3 23 Response surface plots corresponding to the desirability function &hen optimi6ing the follo&ing pair of factors from "A=5 &hile maintaining constant
e remainingt&o at their optimum values: 9A; solvent composition 9 A(8; vs m)phosphoric acid concentrationJ 9E; e*traction time vs temperature3 Results &ere
evaluated using a #
con@dence interval3
reagent
due
to
its
high
ionic
strength
per
unit
concentration
i
n
theaqueous phase3 he lo & recoveries observed for the studied
anti)
biotics
could be e*plained by
the presence
of "g2I5 because
hisdivalent
cation
can
be
chelated
by
quinolones5
as
it
has
been
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previously described G!7H3 he negative e+ect of "gI2 ion
s &a s
especially important for amphoteric quinolones 9Uig3 !A;5
&hich
form
comple*es
more
easily
&ith
divalent
and
trivalent
catio
ns5
in
comparison &ith acidic quinolones 9.IR5 (I85 >V>5 U/,5 and
8A/;3
he determination of the proper amount of salt is also i
mpor)
tant5
since
this
parameter
can
be
used
to
control
the
percentage
of
&ater in the organic phase 9and vice versa;3 Anastassiade
s et al3
G2H stated that the more 8a(l is added to the system5 th
e more
complete the phase separation
becomes3 herefore5
less
&ater
remains in the A(8 phase3 Pater in organic phase enables
a cer)
tain
degree
of
adOustment
in
the
polarity
of
the
phases5
so
th
at
the
e*traction
ef @ciency
of
the
analytes that
are
dissolved
i
nto the
organic layer depends greatly on the applied amount of salt into
the system3 In this conte*t5 it has also to be considered t
hat salt
can
also
produce
the
e*traction
of
polar
co)e*tractives5
decreasing
the
recovery
of
the
analytes3
ome
studies
performed
on
pesti
cides
have also found that the amount of 8a(l used during the
parti)
tioning
had
a
great
inBuence
on
the
pea
shapes
and
areas
of
the
analytes3
hi s
e+ect
is
also
related
to
the
amount
and
nature
of
theco)e*tracted matri*
components G2CH3 "ass of 8a(l from
1
to
4
g
&a s
evaluated5
being
selected
!
g
8a(l
as
optimum
value3
!32323
,se
of
perchloric acid
during
A//=According to a previous &or G!CH and considering that e*tract
on
of quinolones is highly dependent
of p-5 the p- during A//=
&as
also
adOusted
using
-(l>4 $
9&&;3
his
acid
&as
spec
cally
selected due that the chaotropic e+ect e*hibited by inoanic
counter)anions as perchlorate &as found to be the most e+e
tive
under
acidic
conditions3
In
this
study5
the
results
may
also
implyhe
important
role
of
ion)paring
for
the
e*traction
of
quinolones
in
the
salting)out e*traction procedure3 he addition of 7# and 1#
m
of
-(l>4 $ to the "A= e*tract &as evaluated5 &hich produed areduction of p- during A//= from !3 9&ithout acid; to 13# 97
# m/
-(l>4; and 13 91#
m/ -(l>4;3 Phen this acid &as used to a
idify
the
e*tract5
follo&ed
by
liquid0liquid
partitioning
formed
by
addition
of
8a(l5
an
increase
in
the
e*traction
recoveries
&as
observed
foallanalytes &hen p-r13# 9Uig3 !E;3 A volume of 7#
m/ of $
&&;
perchloric
acid
as
compromise
value
&as
selected3
!323!3
>ptimi6ation
of
cleanup
by
a
dispersive
.=
sorbent
After the analytes
&ere partitioned
in the organic
phas
by
A//=5
the
organic
phase
&as
further
cleaned
up
and
dri
d
by
mi*ing
&ith
.=
sorbents
and
anhydrous
"g>43
he
sorbents
&
rechosen
to
retain
the
matri*
components
and
to
enable
the
anal
esof interest
to stay in
the A(8
phase3 radition ally5 a dispersive
.=
cleanup
has
been
carried
out
in
studies
that
employ
'u=(h=R
&o
sorbents
&ere
evaluated:
.A
and
(1C3
.A
acts
lie
a
&
a
anionic
e*changer
and
is
able
to
form
strong
interactions
&ith
ot
er
83 Dorival)MarcQa et al3 alanta 1!C 921#; 24702#7 2#!
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Uig3 !3 >ptimi6ation of the cleanup steps3 9A; ype of salt for salt)assisted liquid0liquid e*tractionJ 9E; volume of -(l>4 $ 9vv;3 =rror bars indicate
standard deviation3
9(; Desirability function for the optimi6ation of cleanup step &ith D).= sorbents: amount of .A vs amount of (1C3 Results &ere evaluated using a #
con@dence interval3
compounds
as
hydrogen
bonds
and
dipole0dipole
forces3
"oreover5
it
sho&s
a
high
chelating
e+ect
due
to
the
presence
of
the
pri
mary
and secondary amines3 he result is the strong retention
of acid
components such as fatty
acids and other polar compounds
in the
matri*
G2 5!H 3
-o&ever5
in
order
to
improve
the
cleanup
proc
ess
of
e*tracts5 especially &hen high content of organic matter an
d fat ispresent5 a mi*ture of .A and (1C is usually applied G!H
3 (1C is
speci@cally
used
for
removal
of
co)e*tracted
fat
and
other
lipophilic
compounds from A(8 e*tracts3 hen5 the optimi6ation
of the
cleanup
&ith
sorbents
&as
focused
on
the
study
of
the
compo
sition
of the sorbents employed3 "i*tures of .A and (1C5
as &ell
as
the
use
of
each
one
separately
&ere
checed3
A
three)level)full
factorial
design
&ith
t&o
parameters
&as
performed3
he
amount
of
"g>4
anhydrous
that
is
applied
during
this
cleanup
phase
as
a
desi
ccant5
in order
to eliminate traces of &ater
&hich
complicate
sample
evaporation
and
concentration
steps5
&as
set
at
$
mg3
he e*perimental
design required 12 runs5
including three
cen)
tral points3 he t&o variables5 evaluated at three levels5 &
ere (1C
amount
95
1#
and
!
mg;5
and
.A
amount
95
1#
and
! mg;3
he
design
matri*
is
sho&n
in
able
!
9supplementary
mate
ial;3
Uig3
!(
sho&s
the
plot
of
the
desirability
function
ver
us
the
amounts of .A and (1C sorbents3 .A resulted the most stati
ically
signi@cant
parameter
for
almost
all
compounds5
&ith
a
positive
e+ect
for
both5
its
lineal
and
quadratic
terms3
hese
results
are
accor
ng
toprevious
&ors5
because
the
use
of
.A
in
the
determin
ion
of
quinolones in similar environmental matrices has been
lready
documented G! 5!!H3 .A demonstrated its ability to se
ectively
remove acidic interferences that are main components of co
mpost5
such as humic and fulvic acids5 as &ell as other organic acids
polar
pigments5 carbohydrates5 sugars and fatty acids &ith h
drogen
bonding
properties
G2CH3
-o&ever5
although
(1C
&as
not
inB
u
ntial
in
most
of
the
cases5
it
had
a
negative
e+ect
in
4
of
the
17
quin
lones5
and an important negative interaction &as observed bet&ee
both
sorbents5 so that the use of (1C &as discarded3 According
to the
results
of
the
design5
!
mg
of
.A
&as
selected
as
the
sorbe
used
in the cleanup step5 &hich &as applied as a mi*ture &ith $
mg
of
2#4 83 Dorival)MarcQa et al3 alanta 1!C 921#; 24702#7
"g>43 he e*traction recoveries ranged from 72 to $ for thestudied
antibiotics
9Uig3
4A;3 !32343
"atri* e+ects
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>ne of the maOor dra&bacs of the use of electrospray
is the
suppression
or
enhancement
of
the
analyte
signal
by
co)
e*tracted
substances of the matri*3 hi s matri*
e+ect &as quanti@ed
for
all
analytes
at
a
concentration
of
1#
ng
g?1
for
each
antibiotic3
"atri*
e+ects &ere evaluated by calculating the percentage of
signal
suppression in the
e*tracts3
he
pea
areas
from the
analysis of
spied compost
e*tracts
&ere compared
&ith the
ones
corre)
sponding to the spied solvent 9mobile phase; at the sam
e con)
centration level3 Average values for signal suppression are
com)
piled in Uig3 4E3 In most cases5 matri* suppression
&a s ob
served3
he suppression ranged from to 475 and signal enhanc
ement
&hich ranged from 1 to 2 for all the acidic quinolones5
">V5
"AR and
=8R &as
also observed3 ignal
enhancement
hasbeen
previously
observed
in
the
determination
of
quinolones
in
sl
udge
and similar matrices G!1H3
,sing a previous &or as a reference G24H5 signal supp
ression
ranged
bet&een
!
and
C
for
the
determination
of
quinolo
nes
in
se&age
sludge3
"atri*
e+ects
have
been
reduced
signi@cantly
i
n
this
&or
for
compost
samples5
&hich
could
be
attributed
to
the
appli)
cation of these ne & alternatives of cleanup steps to "A= e
*tracts3 his di+erence could also be due to the type of matri*5
because
unlie
sludge
that
only
sho&s
signal
suppression5
compost
sa
mples
e*hibit
both5
signal
suppression
and
signal
enhancement3
-o&
ever5
in
both
matrices
there
is
a
clear
di+erence
in
matri*
e+ects
c
aused
by
amphoteric
and acidic quinolones5 signal suppression
is sig)
ni@cantly
lo&er
for
acidic
quinolones
in
sludge
samples5
and
o
n
the
other hand5 acidic quinolones al&ays sho& signal enhancem
ent incompost
samples3
!3!3
"ethod
validation
A
seven)point
matri*)matched
calibration
curve
&as
obtained
for
each studied compound in the lineal dynamic ranges that ar
e indi)
cated in able 23 (alibration curves &ere constructed using a
nalyte
surrogate
pea
area
ratio
versus
concentration
of
analyte3
(inc
ophen
9#
ng
g?1
;
&as
selected
as
surrogate3
=ach
calibration
le
vel
&as
made in triplicate and analy6ed t&ice3 able 2 sho&s the statticaland
the
analytical
parameters
obtained
for
each
studied
antibiot
3
he matri* e+ects &ere also evaluated through the calibr
ion
curves prepared in the compost e*tracts and in the initial m
bile
phase3 tudent]s t)test &as applied in order to compare the
cali)
bration
curves3
Uirst5
the
variances
estimated
as
2y* &ere
com
aredby means of a nedecor]s U)test3 tudent]s t)test sho&ed stattical
di+erences
among
slope
values
for
the
calibration
curves
o
the
target
analytes
and
consequently5
the
e*istence
of
matri*
e+ects
&as
con@rmed5
so
that
it
&as
decided
to
quantify
the
analytes
in
sam
ples
using
the
matri*)matched
calibration
in
all
cases3
he
analytical
method
&as
validated
in
terms
of
linearity5
se
ec)
tivity5 sensitivity and accuracy 9trueness and precision;5 accordi
g
to
the protocols described in the , Uood and Drugs Administra
on
9UDA;
guideline
for
Eioanalytical
"ethod
Falidation
G4H3
/inearity3 he determination coef @cient 9R2
; and the lac)
of)@t
test 9. lof ; &ere evaluated3 he values obtained for R2 ranged f
om3!
to
33
.lof values
&ere 4#
in
all
cases3
herefore5
good
linearity
&as
observed
&ithin
the
concentration
ranges3electivity3 hi s
parameter
&as
demonstrated
by
/(0
""
analysis
of
blans3
A
blan
sample
and
a
spied
blan
sample
&th
the analytes &ere e*tracted and their chromatograms &ere c
m)
pared3 8o interferences &ere observed at the retention time of
he
analytes3
hese @ndings
suggest
that
the
spectrometric
condit
ns
ensure
the
high
selectivity
of
the
methods3
Uig3
#A
sho&s
the
R"
chromatograms
obtained
from
a
spied
sample3
ensitivity3 he limit of detection 9/>D; and quanti@c
ion
9/>'; &ere calculated by taing into consideration the stanard
deviation
of
residuals
9y*;5
the
slope
9b;
of
the
calibration
graphs
and an estimate s obtained by
e*trapolation of the sta
dard
deviation of the blan3 he />D &a s !s and the />' &a s
1s3Uound
limits
of
quanti@cation
ranged
from
3#
to
13#
ng
g?1
3
h
se
results are
also
summari6ed
in
able
23
Accuracy 9precision and trueness;3 he precision of the met
od
in terms of intra) and inter)day variability &as evaluated u
ing
spied compost samples at three concentration levels 9#5 2C
and44
ng
g?1
;
for
each
compound3
.recision
9e*pressed
as
rel
tive
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standard deviation5 RD; &as determined from triplicate s
pied
samples during the same day and in # di+erent days3 he v
lues
obtained are summari6ed in able !3
Uig3 43 9A; =*traction recoveries and 9E; matri* e+ects 9 signal suppression; for the target antibiotics for the &hole optimi6ed e*traction method3 rror bars indicatestandard deviation3
83 Dorival)MarcQa et al3 alanta 1!C 921#; 24702#7 2##
able 2
Analytical and statistical parameters3
.arametersa
">V "AR >U/ =8R />" (I. =8> 8>R .I.
n 42 42 42 42 42 42 42 42 42
b 9g ng?1
; 131
_ 1
?213#
_ 1
?2232
_ 1
?213#
_ 1
?213
_ 1
?2#37
_ 1
?!13$
_ 1
?!37
_ 1
?!23#
_ 1
?!
b 9g ng
?1; $32
_ 1
?#132
_ 1
?413$
_ 1
?473
_ 1
?#437
_ 1
?#!3#
_ 1
?#13#
_ 1
?#23!
_ 1
?423$
_ 1
?#
y* $31 _ 1?!
C3# _ 1?!
C32 _ 1?!
73C _ 1?!
43$ _ 1?!
434 _ 1?!
13# _ 1?!
131 _ 1?2
132 _ 1?!
R2 9; 3CC 37# 3C2 3 32 3C4 3$$ C3C# 3!2
.lof
9; !232 1131 13 !#31 473# 2$37 1132 1132 1C3/>D 9ng g?
1; 3! 3! 32 3! 32 34 3# 34 34
/>' 9ng g?1
; 3 13 37 3 3C 132 13# 13! 13!
/DR 9ng g?1
; 3044 13#0$4 1320#$ 30#$ 3C0#$ 1320#$ 13#044 13C044 13044
.arametersa
DIU AR DA8 .IR (I8 >V> U/, 8A/
n 42 42 42 42 42 42 42 42
b 9g ng?1
; 23$
_ 1
?213!
_ 1
?2232
_ 1
?2#3#
_ 1
?2131
_ 1
?1#3#
_ 1
?2#3!
_ 1
?2$37
_ 1
?2
b 9g ng
?1; 34
_ 1
?##37
_ 1
?#13!
_ 1
?413#
_ 1
?443!
_ 1
?413
_ 1
?413$
_ 1
?4!34
_ 1
?4
y* C3! _ 1?!
#3$ _ 1?!
13! _ 1?2
232 _ 1?2
43! _ 1?2
13 _ 1?2
13$ _ 1?2
434 _ 1?2
R2 9; 3# 32 3C7 37 3# 3$ 37 3CC
.lof 9; 7!3C 473$ 2C31 143$ 7231 C23C 1134 !73/>D 9ng g?
1; 32 32 3! 32 32 32 32 3!
/>' 9ng g?1
; 3$ 37 13 3$ 3$ 3$ 3# 13
/DR 9ng g?1
; 3$044 37044 130#$ 3$0$4 3$0#$ 3$0#$ 3#0#$ 130#$
b^slopeJ b^slope standard deviationJ y*^regression standard deviationJ R2
^determination coef@cientJ J .lof ̂ . value of lac)of)@t testJ />
D^limit of detectionJ
/>'^limit of quanti@cationJ /DR^linear dynamic range3a
n^points of calibration3
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the high levels 9hundreds of ng
g?1
; that &ere found for
these
quinolones could be related &ith their high consumption3
"ore)
over5 =A(]s study also con@rms the continued substantial
use of
quinolones in =urope 9e3g3 ">V;3 >n the other hand5 other study
G42H also reveals the much greater proportion of quinolone
s that
are consumed in pain5 especially by the elderly5 although
these
antibiotics are not considered as @rst)line treatment f
or the
maOority of infections in primary care5 according to panish
guidelines3 Another e*planation for the high concentration
s that
&ere found for some quinolones in the analy6ed compost sa
mples
could be their recogni6ed persistence5 &hich have been a
lready
proved in sludge G4!H5 although there is not
yet enough evi
dence
for this behavior for quinolones in compost3 It is note&orthy
that=8R
and
"AR
&ere
the
only
quinolones
of
e*clusively
veterin
arian
use that &ere found in samples5 &hich con@rm s that =8R
is the
most
used
veterinarian
quinolone
and
the
e*tensive
use
of
"AR in
the
treatment
of
a
&ide
range
of
diseases
in
dogs
and
cats5
&hich
are
the
most
common
pets3
43 (onclusions
he present &or presents an e+ective multi)residue procedure
to determine the content of strong sorbed quinolones in co
mpost
samples3
his
approach
requires
the
use
of
a
drastic
e*tractio
n
pro)
cedure
&hich
&as
carefully
optimi6ed
using
chemometric
strat
egies3
he selected e*traction technique &as "A= at lo & p- value
s pro)
vided by an
e*traction
solvent
containing
m)phosphoric
ac
id3
Ey
9;a 9n^$; 9n^!;
">V # 1!32 23$ 431
2C 143 3C 13$
44 113# 34 134
"AR # 3$ !31 13C
2C 13! 232 13C
44 13$ 32 13!
>U/ # 34 13! 13!
2C C3C 13 23C
44 37 13! 13C
=8R # 3 31 131
2C C3# 131 !34
44
13
3#
37/>"
#
1134
!3
43!
2C 3$ 37 3C
44 37 3$ 13!
(I. # C3# 13 13
2C 13# 31 132
44 123! 234 23$
=8> # #3! 3 $34
2C 737 3! 3C
44 73# 3C !31
8>R # $31 37 37
2C 13! 23$ 13$
44 C3 131 13
.I. # 73 13# 3$
2C 1!31 137 13
44 1132 3 131
DIU # 131 3 23#
2C C3# 32 132
44
3C
34
131
AR # 137 137 132
2C 113$ 131 13
44 13 3! 134
DA8 # 131 3C 134
2C 3# 31 13
44 3 3! 13$
.IR # 13$ 134 13!
2C C3 31 13!
44 34 3! 37
(I8 # 123# 31 13
2C 37 34 13$
44 3! 31 32
>V> # 37 31 132
2C 1132 32 13#
44 3C 32 3
U/, # 131 31 3$
2C
131
31
3444 13# 31 3$
8A/ # 1$3! 3# 131
2C 134 3$ 131
44 C3 34 13!
a RD 9; percentages3
combining a quic and
simple cleanup step
based on A//= an
D)
.= &ith ,-./(0"" analysis5
the obtained
method
is
oth
selective
and
sensitive3
his
procedure
enables
the
determinatioof
target
analytes
at
ng
g?1
levels3
Uinally5
the
method
&as
succesully
validated5 obtaining very lo & />D 9bet&een 32 and 3# ng g?1
;5
igh
e*traction
recoveries
9720$;
and
precision5
besides
the
signi@cant
reduction of matri*
e+ects5
&hich is an important achievement
considering the strong interactions that form quinolones
&ith
matrices &ith high content in organic matter3
hi s method becomes a good alternative to the very fe&
and
inef @cient
e*isting methods5 since it
o+ers besides simplicity
andrapidity
of
operation5
very
good
e*traction
recoveries
for
the
taet
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antibiotics3 he method &as applied to the determination o
f the
levels
of
the
target
antibiotics
in
di+erent
compost
samples
a
nd
it
may
be
used
to
perform
screening
studies
about
the
presence
nd
83 Dorival)MarcQa et al3 alanta 1!C 921#; 24702#7 2#7
able 4
(oncentrations of quinolones 9ng g?1
; in commercial compost samples3
Uound amount in samples 9ng g?1
;a
1 2 ! 4 # $ 7 C
">V 73# 93$; 4!34 934; 1137 931; 32 932; 8D 8D 1!37 93!; #43# 93C;
"AR 8D 8D 8D 8D 1!3 931; 1!3# 932; 8D 8D
>U/ 24$3# 913$; 137 93C; 21#3$ 9232; 23 9234; 7132 913$; $$31 937; 137 9131; 1137 9231;
=8R 2237 9134; $7434 923!; !$3C 937; 1432 934; 73# 9132; 1C3 9131; 3 93!; 243# 932;
(I. $$7 92; #72 92; !2 92; #4$ 92; C!$ 92; 1$7 92; 1#2 92; 27 92;
8>R #C 91; 8D 8D 7 91; 1!1 92; 8D 4! 92; # 91;
.IR 8D 43 931; 8D 8D 13C 931; 8D $31 93!; 132 932;
(I8 3$ 93#; !3 932; 237 937; 13# 931; !3$ 93!; !3C 931; !3# 932; 8D
>V> 37 93!; 3 93$; 1#3$ 9132; $3$ 934; 1C34 913!; 8D 232 93!; 8D
U/, 8D !37 934; 8D 8D 8D 23 93!; 137 932; #3C 93!;
8A/ !3 932; !32 931; 2! 91; 231 93!; 1$3# 9131; 13! 931; 8D C34 93!;
a"ean of $ determinationsJ 8D: not detected 9o/>D;3 tandard deviations are in parentheses3
@nal
fate
of
these
substances
in
the
environment5
as
&ell
as
in
the
study
of
the
ef @ciency
of
the
composting
process
in
the
remo
val
of
this
important
family
of
antibiotics5
taing
into
consideration
that
compost is used on agricultural land5 &here they pose a s
erious
environmental threat3
Acno&ledgements
his study &as supported by the panish "inistry of cience
and
Innovation
9.roOect
no3
('211)2421;3
Appendi* A3 upplementary material
upplementary material associated &ith this article can be
found
in
the
online
version
at
doi:1311$O3talanta321#3!3
113
References
G1H /3 Uin5 I3 Dror5 E3 Eero&it65 (hemosphere C$ 9212; 1440143
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pain5
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noobservatoriodocsantibioticos3pdf ⟩ 9accessed 131314;3
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