improved sample treatment (11)

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



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



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=->

=/;



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



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



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



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



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*



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



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#!



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



>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



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



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



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

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pain5

23 Available from: ⟨http:&&&3aemps3gob3esmedicamentos,so-uma

noobservatoriodocsantibioticos3pdf ⟩ 9accessed 131314;3

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improved sample treatment (11)

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