An overview of sample preparation procedures for LC-MS multiclass antibiotic determination in...

REVIEW

An overview of sample preparation proceduresfor LC-MS multiclass antibiotic determinationin environmental and food samples

María Cruz Moreno-Bondi &María Dolores Marazuela & Sonia Herranz &

Erika Rodriguez

Received: 7 April 2009 /Revised: 10 June 2009 /Accepted: 15 June 2009 /Published online: 25 July 2009# Springer-Verlag 2009

Abstract Antibiotics are a class of pharmaceuticals that areof great interest due to the large volumes of thesesubstances that are consumed in both human and veterinarymedicine, and due to their status as the agents responsiblefor bacterial resistance. They can be present in foodstuffsand in environmental samples as multicomponent chemicalmixtures that exhibit a wide range of mechanisms of action.Moreover, they can be transformed into different metabo-lites by the action of microorganisms, as well as by otherphysical or chemical means, resulting in mixtures withhigher ecotoxicities and risks to human health than those ofthe individual compounds. Therefore, there is growinginterest in the availability of multiclass methods for theanalysis of antimicrobial mixtures in environmental andfood samples at very low concentrations. Liquid chroma-tography (LC) has become the technique of choice formulticlass analysis, especially when coupled to massspectrometry (LC-MS) and tandem MS (LC-MS2). How-ever, due to the complexity of the matrix, in most cases anextraction step for sample clean-up and preconcentration isrequired before analysis in order to achieve the requiredsensitivities. This paper reviews the most recent develop-ments and applications of multiclass antimicrobial determi-nation in environmental and food matrices, emphasizing thepractical aspects of sample preparation for the simultaneousextraction of antimicrobials from the selected samples.Future trends in the application of LC-MS-based techniquesto multiclass antibiotic analysis are also presented.

Keywords Antibiotic monitoring . Environmental analysis .

Food analysis . Extraction . LC-MS

Introduction

Antibiotics are defined as drugs of natural, semisynthetic orsynthetic origin with antibacterial, antifungal or antipara-sitic activity [1]. These drugs are used as chemotherapeuticagents in the treatment of infectious diseases in humans andanimals. They can also be used as growth promoters inlivestock farming (although EU legislation has forbiddenthis practice since January 1, 2006 [2]), as well as inagriculture to control bacterial diseases when growing fruitor vegetables and in bee-keeping [1].

Antibiotics have attracted a great deal of attention duringthe last decade, especially in relation to food safety andtheir presence, behavior and fate in the environment.Antibiotic use is the major contributor to the selection andpropagation of resistant bacterial strains that represent aserious health risk to humans and animals. Resistance tosome antibiotics can emerge after controlled treatment, afterthe prolonged use and/or following the application ofconcentrations that are too low to cure but are high enoughto promote the emergence of resistant bacterial strains.

The main antibiotics used in both human and veterinarymedicine fall into the following classes : β-lactams(β-LCs), tetracyclines (TCs), macrolides (MCs), amino-glycosides (AGs), amphenicols (AMPs), quinolones (Qs)/fluoroquinolones (FQs), sulfonamides (SAs), lincosamides(LCs), glycopeptides (GPs) and polyether ionophores(IPhs) (Fig. 1) [1]. They can be classified according totheir chemical structure or mechanism of action. Antibioticsare used extensively in human and veterinary medicine, as

M. C. Moreno-Bondi (*) :M. D. Marazuela : S. Herranz :E. RodriguezDepartment of Analytical Chemistry, Faculty of Chemistry,Universidad Complutense de Madrid,28040 Madrid, Spaine-mail: [email protected]

Anal Bioanal Chem (2009) 395:921–946DOI 10.1007/s00216-009-2920-8

well as in aquaculture, in order to prevent (prophylaxis) ortreat microbial infections.

These pharmaceuticals can be administered by injection(intravenously, intramuscularly, or subcutaneously), orally,topically on the skin, or by intramammary and intrauterine

infusions in veterinary medicine. All of these routes canlead to the appearance of residues in foods of animal origin,especially when the drugs are used in the wrong or anabusive way (e.g., with withdrawal periods that are tooshort, incorrect doses, self-medication, etc.).

QUINOLONES /FLUOROQUINOLONES

First generation Second generation(FLUOROQUINOLONES)

Third generation(FLUOROQUINOLONES)

N N

O

COOH

H3C

CH2CH3

Nalidixic acid

Y N

OCOOH

R2

R1

F

(Bicyclis)N

OCOOHF

R1

YYCH3 (Tricyclics)

N N

OCOOHO

O

CH2CH3

N

OCOOH

NH

FHN N

ON

CO2HO

F

CH3NH3C

Oxolinic acid Enrofloxacin Ofloxacin

β-LACTAMS

N

S

OCOOH

HNR

O N

R1

S

O N

S

OR2

COOH

HNR1

O NSO3HO

HNR

O

Penicillins Carbapenems Cephalosporins Monobactams

SULFONAMIDES TETRACYCLINES

H2N SHN

O

ON

N 3

CH3

OH O OH O O

NH2

OHH3C OH N(CH3)2

OH

Sulfisomidine Tetracycline

H2N SHN

O

O

N

NCH3

CH3

OH O OH O O

NH2

OHCH3 N(CH3)2

OH

OH

H2N SHN

OR

O

Sulfamethazine

R1

OH O OH O O

NH2

OHR2 R3

R4 N(CH3)2

OH

Doxicycline

AMINOGLYCOSIDES

O O

H2N NH2

OH

O

O

OHHO OH

OH

OH

NH2

HO

NH2

O O

H2N NH2

OH

O

OHO NH2

OH

OH

NH2

HO

NH2

Kanamycin A Tobramycin

COOH

CH

Fig. 1 Chemical structures of the main classes of antibiotics

922 M.C. Moreno-Bondi et al.

Data on antibiotic consumption at international level arescarce and heterogeneous. The total antibiotic consumptionaround the world was estimated at between 100,000 and200,000 tons per year [3]. According to a survey carried out

in 2000 by the European Federation of Animal Health(FEDESA), 13,216 tons of antibiotics were used in the EUand Switzerland in 1999, 65% of which were applied inhuman medicine, 29% in veterinary medicine, and 6% as

O O

H2N NH

OH

O

OHO OH

OH

OH

NH2

HO

NH2

OH

RO

NH2

OHR =

OO O

OO

OH2NH2C

HO

NH2HO

H2N OH

NH2

OH

CH2OHNH2

OH

OHNH2CH2

Amikacin Neomycin

MACROLIDES

R1

ORO

O

HOHO

X

O

R2

O

O

N

OHOCH3

N

O

O

HO

HO

O

O O

O

N

OHOCH3

O

ORO

OH

O

O

N

OCH3

OR1O

O

OR2OR2

OH

Basic structure of 14-membered macrolides Basic structure of 15-membered macrolides Basic structure of 16-membered macrolides

AMPHENICOLS GLYCOPEPTIDES

O2N

OH OH

HN

O

Cl

Cl

Chloramphenicol

LINCOSAMIDES

O

HO

H2N

O

HO OHOH

OO

NH

O

O

HN

NH

HO

HN

HO OH

OH

O

OHN

NH

ONH

OH

NH2

O O

Cl Cl

O

O

HO

HOS

Cl

HNH

O

N

Clindamycin Vancomycin

POLYETHER IONOPHORES

CH3

OH

CH3

OOHCH2CH3

H3C

H3CCOOH

HO

H3C

CH3

OH O

O OH

H3CH2C OO

O

O

OHH3C

OH O

COOH

OCH3

HO

O

O

O

O

CH2CH3

HOH2COH

Lasalocid Salinomycin Monensin

Fig. 1 (continued)

An overview of sample preparation procedures for LC-MS multiclass antibiotic determination in environmental and food samples 923

growth promoters. Between 1997 and 1999, the totalantibiotic consumption increased by about 10%, althoughtheir use as growth promoters decreased by 51% during thisperiod. According to a recent report in the United States,approximately 11,200 metric tons of antibiotics were usedas growth promoters for cattle, hogs and poultry [4].

The European Surveillance of Antimicrobial Consump-tion (ESAC) webpage provides information about thespecific consumption of antibiotics for human medicine inEurope, expressed as “defined daily doses” (DDD, WorldHealth Organisation definition) [5]. There is great variationin the use patterns as well as in the classes of antibioticsused in different countries. Figure 2 summarizes data on theapplication of antimicrobials for systemic use in DDD/100inhabitants/day during 2006 in 24 European countries [5].The group of β-LC antibiotics—especially penicillins(PCs), cephalosporins (CPs) and, to a minor extent,carbapenems (CPNs)—accounts for approximately 50–70% of ambulatory antibiotic consumption in these selectedcountries [5, 6]. The second most important group are theMCs, followed by the TCs, FQs and SAs.

There are significant differences among the differentclasses of antimicrobials in terms of their importance asenvironmental contaminants. β-LCs, the most widely usedantibiotics, are easily hydrolyzed and are not usuallythought to be a serious threat to the environment, butSAs, MCs and FQs are much more stable [7] and have beendetected in different environmental compartments [1].

After consumption, antibiotics are often metabolizedonly partially and are excreted via urine or feces (Fig. 3).The degree of metabolization varies across chemical classesand within a chemical class and depends mainly on the

animal species and mode of application. For instance, thedegrees of metabolism of TCs, MCs and amoxicillin (a β-LC) are lower than 20%, whereas it is higher than 80% forSAs [8, 9]. Antibiotic metabolites can be more toxic tohumans than the parent compounds, although not muchinformation is available on this area [10].

Disposal of unused therapeutic drugs or residues fromthe plants that produce them can also represent a source ofenvironmental contamination [11]. Once they are releasedinto the environment, the nonmetabolized active drug,along with its metabolites, may be degraded, transportedand distributed between different environmental compart-ments. The parent compounds and metabolites are onlypartially eliminated at sewage treatment plants (STPs).Effluents from the STPs are discharged into receivingsurface waters, and trace amounts of these pharmaceuticalshave been detected in surface and ground waters and—more rarely—in drinking water [12].

They can also be present in sediments, especiallybeneath fish farms [1], where antimicrobials are fed directlyinto the water to treat infections in farmed fish. Theapplication of sewage sludge or manure to soils can alsobe a potential route for antibiotics to enter the terrestrialenvironment. The manure and slurry are typically collectedand stored before being applied to the land as fertilizer.During storage, some antibiotics can degrade (particularlySAs, β-LCs, MCs and AGs), whereas others (such as Qsand TCs) are more persistent [13].

Once the drugs reach the soil, they can be partiallyadsorbed onto the soil particles transported to surfacewaters via overland flows or drain flows, leached intogroundwater, and/or degraded. The adsorption of antibiotics

Antibiotic class

Penicillins Other B-Lactams Tetracyclines Macrolides Quinolones Sulfonamides Others

DD

D/1

000

inh

./day

in 2

006

0

50

100

150

200

250Fig. 2 Data on the total use ofantimicrobials for systemic use,expressed in DDD/1000 inhab-itants/day in 2006,corresponding to Austria, Bel-gium, Bulgaria, Croatia, Cyprus,Denmark, Finland, France,Greece, Hungary, Iceland, Ire-land, Israel, Italy, Lithuania,Luxembourg, Netherlands, Nor-way, Portugal, Russia, Slovakia,Slovenia, Spain and Sweden [5]


at the organic and mineral exchange sites in soils is mostlyattributed to charge transfer and ion interactions and not tohydrophobic partitioning [14]. Therefore, sorption will bestrongly influenced by the pH of the medium, which willaffect antibiotic mobility and transport. Antibiotic degrada-tion in the environment can occur through the action ofmicroorganisms, photodegradation processes and/or hydro-lysis [13]. It has been shown that the binding of antibioticsto soil particles or to sediments hampers biodegradation,increasing their persistence in the environment [15]. Forexample, Qs are strongly adsorbed onto sewage sludge,soils and sediments, and some studies have shown that arenot biodegraded in sediments [4]. However, they arerapidly photodegraded in waters, as are TCs, ivermectinand furazolidone, although this process is also less effectivewhen the drugs are bound to the soil surface [15].

The significance of the presence of these compounds atlow levels in the environment is currently unclear, butaccording to EU directives 2001/83/EC [16] and 81/852/EC[17] and their amendments, all new pharmaceuticals mustundergo an environmental risk assessment before they areintroduced into the market. Guidelines on the assessment ofthe environmental impact of veterinary and human pharma-ceuticals have been issued by the European MedicinesEvaluation Agency (EMEA) and by the Food and DrugAdministration (FDA) in the USA [18, 19]. On the otherhand, the International Cooperation on Harmonisation ofTechnical Requirements for Registration of VeterinaryMedicinal Products (VICH) has developed more specificguidelines for veterinary products in an effort to ensure aharmonized approach for Japan, the USA, and the EU [20,21].

Concentration limits or tolerance levels of antibiotics inthe environment are not regulated, but they vary betweenthe higher µg/L range in hospital effluents, the lower µg/Lrange in municipal wastewater, and the higher and lowerµg/L ranges in different surface waters, groundwater andseawater [1].

Regarding foodstuffs, the FDA in the USA and the EUhave set maximum residue limits (MRLs) for veterinarydrug residues in foods of animal origin to ensure consumersafety [22–24]. However, legislation can differ considerablybetween different countries, especially in developingregions, where, in many cases, national food regulationsare still being created and updated [25]. Moreover, MRLsare not established for all of the antibiotics found in eachfood commodity and food-producing species. Therefore,veterinary drug-residue control represents an importantissue in ensuring consumer protection.

As mentioned before, antibiotic residues do not occur asisolated parent drugs in the environment. They can betransformed into different metabolites by the action ofmicroorganisms, as well as by other physical or chemicalmeans. On the other hand, a broad range of pharmaceuticalsare applied for human and veterinary uses, so they will bepresent in the environment as multicomponent chemicalmixtures with a wide range of different mechanisms ofaction. This “cocktail” usually has a synergic effect,showing a higher ecotoxicity than the individual com-pounds, as some authors have reported [26, 27]. Accordingto the current guidelines for medical product authorizationissued by EMEA, ecotoxicological assessment is notnecessary if the predicted environmental concentrations(PEC) of human pharmaceuticals in the aquatic environ-

DomesticWaste

Excretion(Private households)

Excretion(Hospitals)

Waste Disposal(unused pharmaceuticals)

Antibiotics forhuman use

Ground Water

Drinking Water

SewageSludge

Municipal Waste Water

Sewage Treatment Plant(STP)

Landfill

Antibiotics forveterinary use

Excretion

Soil

Manure

Surface Water

Antibioticproduction

plants

Aquaculture

Faecal Shedding

Field Greenland

Growthpromoters

AlimentaryCycle

Fig. 3 Sources and pathwaysfor antibiotic residues in theenvironment (adapted from [6])


ment are below 0.01 μg L−1 or below 10 μg kg−1 and0.1 μg L−1 in soil and groundwater, respectively, in the caseof veterinary medicines. Obviously, these cut-off values arenot sufficient for mixtures of substances with similar oreven dissimilar activities [28]. Therefore, there is growinginterest in the availability of multiclass methods for theanalysis of pharmaceutical mixtures in the various environ-mental compartments that allow realistic evaluation of theecotoxicological effects of these drugs for different organ-isms. On the other hand, they are also requested byregulatory agencies as part of their requirement to monitorthe food supply for regulated veterinary residues andcontaminants. Moreover, the application of multiresidueanalysis methods also allows the time and cost of analysisto be reduced.

Antibiotics, and in general pharmaceutical compounds,in contrast to chemical industrial pollutants, possess specialcharacteristics that make multiclass analysis difficult. Theyare comparatively large and chemically complex molecules,with several functionalities and multiple ionization siteswithin the same molecule. They can therefore be cationic,anionic or zwitterionic, and the solution pH can affect theirphysicochemical properties, sorption behaviors, photo-stabilities, as well as their antimicrobial activities andtoxicities [29]. The diversity of the chemical properties ofdifferent antibiotics, and the low concentration levels atwhich they can be present in samples, increase the difficultyinvolved in finding generic analytical procedures formulticlass analysis, especially for complex matrices suchas food or sludge.

The simultaneous analysis of different antimicrobialsrequires chromatographic separation using gas (GC) orliquid chromatography (LC). The first has found limitedapplicability in antibiotic analysis. Due to the polar nature,low volatility and thermal stability of these drugs, theyrequire derivatization before analysis. Therefore, LC hasbecome the technique of choice for multiclass analysis,especially when coupled to mass spectrometry (MS) andtandem MS (LC-MS2), the latter allowing increasedsensitivity and selectivity in complex matrices [30, 31].Despite these characteristics, due to the low concentrationlevels of antibiotics found in most environmental [32] andfood [33] samples, the majority of applications require aextraction step, for both sample clean-up and preconcentra-tion, before analysis.

In this review we present an overview of recentdevelopments in the analysis of multiclass antibioticresidues in environmental and food samples. Most of thereferences included herein correspond to methods publishedin the last five years for the analysis of more than twoantimicrobial classes. We discuss the main sample treat-ment methods, as well as the analytical procedures reportedfor multiresidue determination in the selected matrices,

emphasizing their analytical characteristics and futureprospects.

Analytical methodology

Sample preparation

The aim of sample preparation procedures for single-classresidue analysis is to obtain the maximum recoveries of theanalytes in a given matrix. In contrast, during thedevelopment of multiclass methods, recovery optimizationremains an important but secondary issue, because the maingoal is the simultaneous extraction of as many analytesfrom multiple classes as possible. Obviously, the morevariation there is in their physicochemical properties (e.g.,pKa, polarity, solubility, stability, etc.), the greater thedifficulty in finding a generic extraction procedure for allanalytes with acceptable recoveries of them. In addition, thecomplexity of some matrices, especially when consideringfood samples, requires clean-up steps or dilution of theextracts prior to LC-MS determination in order to avoidmatrix effects [30]. Thus, up to now, multiclass methodshave been relatively scarce compared to multimatrix orsingle-class residue methods. However, the situation ischanging rapidly, and the availability of straightforwardmulticlass extraction procedures is increasing in theliterature due to the interest in applying them to routinemonitoring programs that would drastically reduce the timeand effort devoted to sample preparation. Wheneverpossible, sample preparation procedures for multiclassanalysis should be as simple as possible in order to achievehigh sample throughput.

Environmental matrices

Multiclass antibiotic analysis has mainly been carried out inenvironmental water samples and, to a lesser extent, insludge and soil, which are much more complex matrices.Sample treatment methods for liquid samples (e.g., surfacewater, groundwater or drinking water) are usually based onsolid-phase extraction (SPE) using polymeric sorbents,although other techniques such as lyophilization or liquid–liquid extraction (LLE) have also been reported [34, 35].Prior to SPE, the samples are filtered to remove particles insuspension. The relatively low antibiotic concentrationspredicted in environmental waters require preconcentrationfactors that are typically of the order of 1000, and samplevolumes are usually in the range 100–1000 mL [30].Tables 1 and 2 summarize the sample preparation proce-dures as well as the analytical methods available for thedetermination of multiclass antibiotics in environmentalsamples.


Tab

le1

Sum

maryof

metho

dsused

fortheextractio

nof

antib

ioticsin

environm

entalsamples

Com

pounds

Matrix

Pretreatm

ent

Treatment/S

PE

Recoveries(%

)References

7SAs,3MCs,

7FQs,6TCs,

TMP

CDW

Addition

ofascorbic

acid,25

mg/L

Sam

plepH

3.0.

86-125%

forMCs,FQs.

TMPandmostTCs

[12]

Filtratio

n(0.45µgnylonfilters)

Offlin

eSPE:OasisHLB

Addition

4mL2.5g/L

Na 2EDTA

perlitre

ofsample

Sam

plevolume500mL

3SAs,TMP,

OTC,

ENRO,PEN

GWW

Filtratio

n(W

hatm

anfilterpaper+

0.45

µm

nylonmem

branefilter)

Sam

plepH

4.0.

Offlin

eSPE:OasisHBL.

Sam

plevolume:

100mL.Preconcentration:

×100

11.2–9

7.9%

;RSD≤1

0.1%

[36]

4MCs,6SAs+

NAc-SMX,TMP

EFFL

Filtratio

n(0.45µm

cellu

lose

nitratefilters).Addition

of1gNaC

lSam

plepH

4.0(H

2SO4).Offlin

eSPE:OasisHLB.

Sam

plevolume:

50mL(1°EFFL);250mL

(2°and3°

EFFL).Concentratio

ns:×25

and×500,

respectiv

ely

78–1

24%

except

TMP

(30–

47%);RSD<15%

[37]

5SAs,4MCs,

3FQs,TMP

WW,RW

Filtratio

n(2.7

µm

glassfiberfilterGF/D)

Sam

plepH

3.0(formic

acid).Offlin

eSPE:OasisHLB.

Sam

plevolume:

Raw

WW,100mL;WW

effluent,

200mLandRW,500mL

RW:72

–116%.Secondary

effluent:49

–119%.Primary

effluent:53

–112%;RSD≤1

1%

[38]

3SAs,2FQs,

3TCs,1LCs,

3MCs,TMP

SW,GW,WW

Filtratio

n(1.2

µm

filter+0.2µm

filter).

Addition

of2mL5%

EDTA

Sam

plepH

3.0(40%

orthophosphoricacid).

Offlin

eSPE:OasisHLB.Sam

plevolume:

500mL.

Concentratio

n:×500

SW:74

–129%;RSD≤1

3%.

GW:51

–120%;RSD≤1

1%.

WW:75

–126%;RSD≤2

3%

[39]

CAP,

OFLO,2SAs

WW,GW,

RW,TW

Centrifuge1500

rpm,15

min

filtration

(0.7

µm,47

mm

i.d.glass

microfiberfilter)

Sam

plepH

3.0(H

Cl).Offlin

eSPE:OasisHLB.

Sam

plevolume:

100/250mL(spikedsamples/W

W)

64–9

9%;RSD≤9

%[42]

3SAs,TMP,

3FQs,

CAP

Urban

waters

Addition

ofsodium

azide(0.5

gL−1).

Filtratio

n(glass

fiberfilter)

Addition

ofsodium

chloride

(2.93g/L).

Sam

plepH

4.2(H

Cl).Offlin

eSPE:HLBcartridge.

Sam

plevolume:

350–1000

mL

WWE:63

–126%;RSD≤1

2%.

WWI:65

–112%;RSD≤3

0%[43]

ORN,TMP,

2SAs,

13FQs

RW

Filtratio

n(0.47µm

glassfiberfilter)

Sam

plepH

7.0(orthophosphoric

acid

25%).

Offlin

eSPE:OasisHBL.Sam

plevolume:


×200

70–1

20%;RSD<15%

[44]

3MCs,6FQs+

2QdN

Os,16

SAs,

4TCs

WW

Filtratio

n(0.1

µm

glass

microfiberfilters)

Offlin

eSPE:OasisHLBcartridges.

Sam

plevolume:

1000

mL.Concentratio

n:×1000.

Methoda(m

acrolid

es):samplepH

6.0(3.0

MH2SO4).

Methodb(allothers):samplepH

3.0(3.0

MH2SO4)+

0.5gEDTA

72–9

9%;RSD≤1

0%[46]

1PC,2FQs,6MCs,

1LCs,1SA,1TC

(+oP

s)

STPs

Filtratio

n(glass

microfiberfilterGF/D,

2.7µm)

Offlin

eSPE:a)

samplepH

2(37%

HCl)+

EDTA

+OASIS

MCX;b)

samplepH

7(30%

NH4OH)+

LiChrolut

EN.Sam

plevolume:

500mL.

Preconcentration:

×5000

31–1

31%;RSD≤7

.6%

[47]

ERY,CAP,

SMX

(+oP

s)SW,GW,DW

Filtratio

nSam

plepH

3.0(H

Cl).Offlin

eSPE:OasisMCX.

Sam

plevolume:


×200

63–9

6%;RSD≤1

6%[48]

SMX,CAP(+

oPs)

SW

Filtratio

n(0.7

µm

glassfiberfilterGF/F)

Sam

plepH

2.0(31%

HCl).Offlin

eSPE:OasisMCX.

Sam

plevolume:

1L

SMX:27.2%;RSD

4.5%

.CAP:37.0%;RSD

1.4%

[49]

TMP,

SMX,AMOX,

CAP,

ERY,MET

(+oP

s)

SW,WW

Filtratio

n:SW,0.7µm

glassfiberfilter

GF/F

WW,GF/D

2.7µm

glassfiber

filter+0.7µm

glassfiber

filterGF/F

Sam

plepH

2.0(31%

HCl).Offlin

eSPE:OasisMCX.

Sam

plevolume:

SW,1000

mL;WW,250mL.

Concentratio

ns:×2000

and×500,

respectiv

ely

Ultrapurewater:35

–104%.

SW:27

–84%

.WWE:7–

76%.

WWI:4–

69%;RSD≤1

2%

[50]

3TCs,4SAs,TMP,

2LCs,4MCs,

2PCs,4FQs

(+oP

s)

SW

Filtratio

n(0.7

µm

TCLPglassfiber

filter):250mL+0.2gNa 2EDTA

+H2SO4or

NH4OH

(pH

5.0)

Offlin

eSPE:Strata–X.Sam

plevolume:

250mL

51–1

09%;RSD≤2

4%[51]

SMX,NAc-SMX,

TMP,

ERY

(+oP

s)WWESW

Filtratio

n(0.45µm

glassfiber

filterGFC)

Sam

plepH

3(H

Cl).Offlin

eSPE:Phenomenex

StrataX

56–1

23%;RSD≤2

0%[52]

10FQs,6PCs

GW,SW

Centrifuge3500

rpm,5min

Sam

plepH

2.5(autom

ated

acidification,

700µL10%

GW:57

–123%;RSD≤1

1%.

[53]


Tab

le1

(con

tinued)

Com

pounds

Matrix

Pretreatm

ent

Treatment/S

PE

Recoveries(%

)References

(ifsuspendedparticulatematteris

visible),200µLNH4COOH

500mM/12mLsample

HCOOH):a)

on-lineSPE:C18,samplevolume:

9.8mL;b)

offlineSPE

SW:34

–117%;RSD≤2

4%

3FQs,1CE,2PCs,

1SA,1NTI,1TC,

TMP

Sew

agewater

Filtratio

n(0.45µm

MFmem

branefilter)

Sam

plepH

3(H

2SO4).Offlin

eSPE:layeredC2/ENV

+samplevolume200–

500mL.Concentratio

n:×200–

500)

48–9

0%;RSD≤1

2%[54]

4FQs,3SAs,TMP

WW

Addition

of2mg/LNa 2S2O3.

Filtratio

n(0.5

µm

glassfiberfilter).

Addition

of0.1M

NaC

l

Sam

plepH

2.5(H

3PO4).Offlin

eSPE:anion-exchange

Isolute+OasisHLB.Sam

plevolume:

1000

mL.

Concentratio

n:×1000

Deionized

water:84

–102%.

Secondary

effluent:37

–124%.

Final

effluent:64

–114%;

RSD

10–5

6%

[55]

3MCs,2SAs,

4TCs,7PCs,

TMP,

CAP

Water

Filtratio

nwith

0.45

µm

glassfiberfilter

Methoda:

lyophilization:

1mg/mLEDTA

;methodb:

offlineSPE(excep

tetracyclin

es),samplepH

3.0

(H2SO4),LichroluteEN

+LichroluteC18.Sam

ple

volume:

1000

mL.Concentratio

n:×1000)

Lyophilizatio

n:54

–108%.

SPE:15

–120%;RSD≤1

5%[56]

4TCs,8MCs,

3SAs,6FQs,

TMP,

CAP,

MET,

NIF

(+oP

s)

Environmental

waters

Filtratio

n(1

µm

glassfiberfilters

+0.45

µm

nylonmem

branefilters)

Offlin

eSPE:OasisHLB

SW

54–1

35%

[58]

Sam

ple:

500mL(SW);200mL(W

WE);

100mL(influentwastewater,WWI)

WWE31–1

21%

Addition

of5%

EDTA

(0.1%

inthesample)

WWI21

–151%

10mLSW

RSD

≤25%

4mLWWE

2mLWWI

TMP,

MET,

ERY

(+oP

s)Hospital

WWEs

Filtratio

n(0.7

µm

glassfiberfilter)

Sam

plepH

7.0(2

MH2SO4).Offlin

eSPE:OasisHBL.

Sam

plevolume:


×100

TMP:87.9%;RSD

1.4%

.MET:83.8%;RSD

4.9%

.ERY:95.2%;RSD

2.4%

[59]

2MCs,SMX,TMP,

OFLO

(+oP

s)SW,WWI,

WWE

Filtratio

n(1

µm

glassfiberfilter+

0.45

µm

nylonmem

branefilter)

NosamplepH

adjustment.Offlin

eSPE:OasisHBL.

Sam

plevolume:

500mL(SW);200mL

(effluent);100mL(influent).Preconcentrations:

×500,

×200,

×100,

respectiv

ely

SW:30

–108%;RSD≤9

%.

WWE:30

–116%;RSD≤1

5%.

WWI:40

–111%;RSD≤9

%

[60]

ERY,AZI,SMX,

TMP,

OFLO

(+oP

s)

GW,RW,

WWI,WWE

Filtratio

n(1

µm

glassfiberfilter+

0.45

µm

nylonmem

branefilter)

Offlin

eSPE:OasisHLB.Sam

plevolume:

500mLground

andRW;200mLeffluent;

100mLinfluent.Preconcentrations:×500,

×200and×100,

respectiv

ely

Not

clearlyreported

[61]

13FQs,32

SAs,

12PCs,19

MCs,

5TCs(+

other

veterinary

drugs)

Urine

Sam

pledilutio

n1:10

with

5%acetonitrile

–4–

377%

[62]

3MCs,3SAs,

TMP(+

oPs)

River

sediment

Autoclave.USE50

gsample

Offlin

eSPE:LichroluteEN

+LichroluteC18

Absoluterecovery:

[63]

2x45

mLMeO

H41

–82%

+45

mLacetone

RSD

≤16%

+45

mLethylacetate

Relativerecovery:

Rotary-evaporationat

40°C

,150-200mbar

71–1

21%

RSD

≤23%

6TCs,5SAs

WWI,WWE

Centrifugationat

3000

rpm

×40

min,

4°C

(influent).Filtratio

nwith

0.4µm

glassfiberfilter.Addition

of1mL

5%EDTA

+30

mL0.1M

citric

acid

Sam

plepH

<3.0(40%

H2SO4).Offlin

eSPE:Oasis

HLB.Sam

plevolume:

120mL.

Concentratio

n:×1000

Deionized

water:94.6–101%;

RSD≤1

0.4%

.WWI:77.9–

99.8%;RSD

≤13.9%.

WWE:83.6–1

03.5%;

RSD

≤13.4%

[64]


Tab

le1

(con

tinued)

Com

pounds

Matrix

Pretreatm

ent

Treatment/S

PE

Recoveries(%

)References

5SAs,TMP,

3MCs,

OME,RAN

Sludge

Lyophilizatio

nof

sample

PLE:5gsample+alum

inium

oxide;

water

(pH

3)–M

eOH

(1:1,v/v),1500

psi,

80°C

,5min

preheatin

g,60%

flushvolume,

120s

nitrogen

purgeand1extractio

ncycle(5

min)

54–9

5%;RSD≤1

5%[66]

3MCs,4SAs,TMP

Activate

sludge

1.Lyophilisatio

nOfflin

eSPE:OasisHLB

Absoluterecovery:41

–95%

[69]

Digested

sludge

2.Grind

inamortar

RSD

≤6%

3.PLE

200mgsample+quartz

sand;

MeO

H/H

2O

(1:1,v/v),100bar

(1450psi),100°C

,5min

preheatin

gtim

eand5min

static

time,

120%

flush

volumeand3extractio

ncycles

(5min

percycle),60

snitrogen

purge

4.Dilu

tedsludge

extract.

(Sam

plepH

4or

7)

9SAs,2PCs

Sludge

Lyophilizatio

nof

samplePLE:

5gsample+1gNa 2EDTA

+Hydromatrix;

acetone–MeO

H(1:1,v/v),1500

psi,75

°C,

60%

flushvolumeand3extractio

ncycles

Evaporatio

nof

PLEextractsundernitrogen

stream

.Reconstitu

tioninto

MeO

H–H

2O

(1:19,

v/v).

Offlin

eSPE:OasisHLB

1–85%;RSD<54%

[70]

TMP,

SMX,

TRI,(+oP

s)Soils,digested

sludge

Lyophilizatio

nof

samplePLE:2.7gsample+analytical

grade

seasand;MeO

H–H

2O

(1:1,v/v),1500

psi,

60°C

,5min

heat

time,

100%

flushvolume

and2extractio

ncycles

(5min

percycle),

60snitrogen

purge

Sam

plepH

5.5(formic

acid

oram

monia

solutio

ns).

Offlin

eSPE:WatersOasisHLB

85–1

30%;RSD<13%

[71]

2TCs,1SA,

2MCs

Agricultural

soils

Airdried.

Offlin

eSPE:SAX

andHLBin

tandem

Sandy

soil

[72]

PLE:10

gsample+10

gOttawasand;

MeO

H/citric

acid

0.2M,

pH4.7(1:1,v/v);1500

psi;RT;

10min

preheatin

g;1extractio

ncycle

45.4–1

34.4%

RSD

≤67%

Loamysand

soil

31.3–1

26.6%

RSD

≤32%

4TCs,2FQs

Wellwater,

RW,STP

Filtratio

n:STPsamples:Rundfilter

MN

615filterpaper+0.45

µm

mem

brane

filter.River

andwellsamples:

0.45

µm

mem

branefilter

Sam

plepH

2.8(H

Cl).Offlin

eSPE:OasisHLB.

Sam

plevolume:

WWI,100mL;WWE,250mL;

RW

andwellwater,1000

mL.Concentratio

ns:

×100,

×250and×1000,respectiv

ely

RW:88

–112%.Wellwater:41

–87%

.WWE:68

–89%

.WWI:73

–103%

;RSD<15%

[73]

5TCs,6SAs

GW,SW

Filtratio

n(0.7

µm

glassfiberfilter).

Addition

of75

µL

40%

H2SO4+1mLscoop

ofEDTA

Sam

plepH

2.5.

Offlin

eSPE:HLBWaters.

Sam

plevolume:

123mL

83.8–1

30%

(except

minocyclin

e);

RSD<20%

[74]

ERY,SMX,

TMP,

TRI(+oP

s,endocrinedisruptors

andPCPs)

Water

Sam

plepH

2.0(H

2SO4).Offlin

eSPE:HLBWaters.

Sam

plevolume:

1000

mL.Concentratio

n:×1000

71–9

1%;RSD≤1

7%[75]

5SAs,5TCs,

7MCs,TMP,

CAP,

CAR,(+oP

s)

Water

Addition

of2gNa 2EDTA

Sam

plepH

8.2(H

2SO4or

NaO

Hsolutio

n).

Offlin

eSPE:HBLWaters.Sam

plevolume:

400mL.

Concentratio

n:×800

48–1

50%;RSD≤1

5%[76]

TMP,

1SA,FLOR,

OXO

SW

Centrifugationfor5min

at3000×g

Sam

plepH

5.0–5.2(H

3PO4).Offlin

eSPE:OasisHLB.

Sam

plevolume:

40mL.Concentratio

n:×80

78–9

6%;RSD<11%

[77]

AMOX,TMP,

SMX

(+oP

s)SW,GW

Filtratio

n(0.7

µm

GF/F

glassfiberfilter)

Offlin

eSPE:OasisHLB.Sam

plevolume:

1000

mL.

Concentratio

n:×1000

8–123%

;RSD<21%

[78]

6TCs,6SAs,3MCs

RW

Water

samples:

Offlin

eSPE:OasisHLB.

RW:TCs:100–

127%

[79]

Filtratio

n(0.2-µm

glassfiberfilter)

SAs:76

–124%


Tab

le1

(con

tinued)

Com

pounds

Matrix

Pretreatm

ent

Treatment/S

PE

Recoveries(%

)References

pHsample2.0–

2.5(TCsandSAs),

5.0(M

Ls)

Sam

plevolume:

120mLConcentratio

n(x1000)

MLs:89

–114%

Sedim

ent:

Sedim

ent:

Air-dried

inthedark.Passedthrough

2mm

and75

µm

sieves

TCs:40

–114%

SAs:62

–11%

MLs:53

–128%

3TCs,3SAs,

2FQs,3MCs,TMP

Urban

WW

500mgNa 2EDTA

+5mL10%

Na 2SO3

(per

1000

mLsample)

Sam

plepH

2.8-3.0.

OnlineSPE:OasisHLB.

Deionized

water:

[80]

Filtratio

n(0.45µLacetatecellu

lose

filter)

Sam

plevolume50

mL

94–1

20%

RSD

≤25%

WW

(effluent):

40–9

2%

RSD

≤17%

13FQs,2SAs,

ORN,TMP

MW,SW

Filtratio

n(0.45µm

glassfiberfilter)

Sam

plepH

7.70

–130%

[81]

Offlin

eSPE:OasisHLB

RSD

≤20%

4SAs,4FQs,

4TCs,CAP

GW,LW

,WWI,

WWE

Filtratio

n(1

mm

filter+0.45

µm

glass

fiberfilter)

Sam

plepH

4.0.

GW:76.9–115.1%

[82]

Addition

of1%

form

aldehyde

Offlin

eSPE:OasisHLB

LW:68.6–9

2.6%

Addition

of5mL5%

(w/v)EDTA

Sam

plevolume:

WWE:62.5–9

3.1%

GW:500mL;LW

:250mL;piggerywastewater:50

mL

WWI:58.4–9

8.7%

RSD

≤10%

AMOX,am

oxicillin;AS,

agricultu

ralsoils;AZI,

azith

romycin;CAP,

chloramph

enicol;CAR,carbadox

;CDW,chlorinateddrinking

water;CEs,

ceph

alospo

rines;

DW,drinking

water;EFFL,

effluents

ofmun

icipal

wastewater

treatm

entplants;ENRO,enroflox

acin;ESI-M

S2,electrospray

ionizatio

n–tand

emmass

spectrom

etry;ERY,

erythrom

ycin;FLOR,florfenicol;

FQs,

fluo

roqu

inolon

es;GW,grou

ndwater;HLB,hy

drop

hilic–lipop

hilic

balance;

LCs,

lincosamides;LW

,lake

water;MCs,

macrolid

es;MET,

metronidazole;MW,mineral

water;NAc-SM

X,N-acetyl

sulfam

etho

xazole;NIF,nifuroxazide;NMZs,nitroimidazoles;NTIs,nitroimidazoles;OFLO,ofloxacin;

OME,om

eprazole;oP

ss,otherph

armaceuticals;ORN,ornidazole;OTC,ox

ytetracycline;

OXO,ox

olinic

acid;PCs,penicillins;PENG,penicillinG;PCPs,person

alcare

prod

ucts;PLE,pressurizedliq

uidextractio

n;QdN

Os,qu

inox

alinediox

ides;Qs,qu

inolon

es;RAN,ranitid

ine;

RW,

riverwater;SA

s,sulfon

amides;SM

X,sulfam

etho

xazole;SP

E,solid

-phase

extractio

n;ST

Ps,

sewagetreatm

entplants;SW

,surfacewater;TCs,

tetracyclin

es;TFA

,trifluoroacetic

acid;TMP,

trim

etho

prim

;TRI,triclosan;

TW,tapwater;USE

,ultrasou

nd-assistedsolventextractio

n;WW,wastewater;WWE,effluent

WW;WWI,influent

WW


a) Waste water, surface water and groundwater

Babic et al. [36] have described the preconcentration ofthree SAs, a sulfonamide synergist, trimethoprim (TMP), aTC, a FQ and a β-LC from wastewater of a pharmaceuticalcompany using Oasis HLB cartridges. Recoveries ofbetween 89.3% and 97.9% were obtained, except forpenicillin G/procaine (68.3%) and sulfaguanidine (11.2%).Reported intraday and interday reproducibilities were lowerthan 8.4% and 10.1%, respectively.

Göbel et al. [37] applied HLB sorbents for the extractionof four MC antibiotics, six SAs, the human metabolite ofsulfamethoxazole, N4-acetylsulfametoxazole, and TMP inprimary effluents (1° EFFL, after mechanical treatment),secondary effluents (2° EFFL, after biological treatment), andtertiary effluents (3° EFFL, after sand filtration). The mostinfluential parameter for sample extraction was pH, whichhad a significant impact on the retention of the compounds,especially on SAs; due to their amino groups, SAs showedtheir highest recoveries at pH 4, whereas the recoveries ofMCs and TMP showed almost no pH dependence. Underthese conditions, the metabolite N4-acetylsulfametoxazolewas stable during sample treatment, but erythromycin wasunstable, transforming into erythromycin-H2O. Recoveries ofthe antibiotics in the different sample matrices were above80% (RSD≤18%), except for TMP (30–47%), due to the useof a nonideal surrogate sulfamethazine-phenyl-13C6

(13C6SMZ) for its determination.Senta et al. [38] compared the performances of three

popular extraction cartridges: Envi C18, a classical lipo-philic sorbent, and two polymeric SPE phases, Oasis HLBand Strata X, for the extraction of three classes ofantimicrobials, including six SAs, TMP, three FQs and fiveMCs. Higher recoveries were achieved with the polymericcartridges (>66%), illustrating the importance of thehydrophilic groups in the retention mechanism. In compar-ison to the protocol described by Göbel et al. [37], theoptimized retention pH for all of the antibiotic classes was3.0, and on this occasion it was also crucial to the retentionof FQs. The use of basic conditions (1% NH3 in methanol)during the elution step allowed higher extraction recoveries,especially for MC antibiotics.

Batt et al. [39] developed an SPE-LC-MS2 method forthe analysis of 13 antibiotics and caffeine in surface watersand wastewaters. The selected compounds were represen-tative of different classes used in both human andveterinary medicine, including TCs, FQs, SAs, LCs, andMC antibiotics, which are expected to have differentenvironmental fates and effects. Caffeine monitoring wasalso included, as it behaves as an anthropogenic marker ofuntreated domestic wastewater contamination, and itspresence in surface waters is indicative of untreated sewageoverflows. The antibiotics were preconcentrated using

Oasis HLB cartridges, as in the previous papers, but thesample pH was adjusted to between 2.8 and 3.0 to increasethe retention of the TCs and FQs, which decreases atneutral pH. However, below pH 7.0 erythromycin isimmediately degraded to erythromycin-H2O, which isquantified in the extracts instead of the parent molecule.The recoveries of MC antibiotics were increased by addinga chelating agent, EDTA, that binds to the soluble metals,increasing the antibiotic extraction efficiency. The elutingsolvent was ACN instead of MeOH, as it allowed improvedrecoveries of erythromycin-H2O and roxithromycin. Theoptimized SPE-LC-MS2 procedure was applied to theextraction of these compounds from three different matrices(groundwater, surface water and wastewater), and theanalysis reported the presence of clindamycin (1.1µg L−1)in surface water from the Niagara River (US) and multipleantibiotics (0.10–1.3µg L−1) in wastewater effluents fromtwo different STPs.

Ye et al. [40] reported a multirun analytical method forthe trace determination of 24 antibiotics, including sevenSAs, three MCs, seven Qs/FQs, six TCs, and TMP, inchlorinated drinking water using SPE followed by LC-MS2

analysis. Some antibiotics, sulfamethoxazole, ciprofloxacin,enrofloxacin and erythromycin, can react with free chlorineand are expected to undergo substantial transformationsunder extended exposure to residual chlorine in finisheddrinking water [41]. Ascorbic acid was added to thesamples to increase the storage time, as it quenches residualchlorine without affecting the analysis and stability of theantimicrobials. Preconcentration was carried out in the HLBcartridges with sample loading at pH 3.0, and elution inacidified methanol (0.1% formic acid) to increase therecovery of the piperazinylic quinolones. All antibioticswere successfully extracted from the drinking watersamples with recoveries ranging from 90 to 106%.However, TC and SA losses were observed after solventevaporation before the chromatographic analysis. The TClosses were attributed to antibiotic sorption on the glasssurfaces, but for the SAs, the lower recoveries wereexplained by their possible association with matrix compo-nents that precipitate during solvent reduction and areremoved by filtration before analysis.

HLB cartridges have also been applied in several studiesto evaluate the occurrence and fate of multiclass antibioticsin environmental water samples in several countries. Forexample, the occurrence of SAs, FQs, chloramphenicol(CAP) [42] and TMP [43] in urban river water andwastewater in Guangzhou, China, was evaluated by SPEfollowed by LC-DAD/FLD, indicating that activated sludgetreatment is effective at and necessary for removingantimicrobial substances from municipal sewage.

These cartridges have also been applied to the analysisof 17 antibiotics, belonging to four groups, Qs, SAs, NMZs


Tab

le2

Sum

maryof

multiclass

LC-M

Smetho

dsused

forantib

iotic

residu

eanalysisin

environm

entalsamples

Com

pounds

Matrix

LC

MS

Sensitiv

ityReferences

Colum

nMobile

phase

Type

Ionizatio

nLOD

LOQ

7SAs,3MCs,7FQs,

6TCs,TMP

CDW

PursuitC-18(150

×2

mm,3µm)

Formic

acid

andACN

(gradient)

QqQ

ESI(+)

0.5–

6ng

L−1

1-36

ngL−1

[12]

3SAs,TMP,

OTC,

ENRO,PEN

GWW

Lichrosphere100CN

(125

×4.0

mm,5µm)

10mM

oxalic

acid

andACN

(gradient)

0.1–

40µgL

−11.5–100µg

L−1

[36]

4MCs,6SAs+NAc-

SMX,TMP

EFFL

YMCPro

C18

(150

×2

mm,3µm)

1%form

icacid

andMeO

H(gradient)

QqQ

ESI(+)

3–214

ngL−1

[37]

5SAs,4MCs,3FQs,

TMP

WW,RW

YMCProC18

(150

×2.1

mm,3µm)

0.1%

form

icacid

andMeO

H(gradient)

QqQ

ESI(+)

1–13.2

ngL−1

[38]

3SAs,2FQs,3TCs,

1LCs,3MCs,TMP

SW,GW,WW

BetaB

asic-18C18

(100

×2.1

mm,3µm)

0.3%

form

icacid,MeO

HandACN

(gradient)

ITESI(+)

27–1

90ng

L−1

100–650

ngL−1

[39]

CAP,

OFLO,2SAs

WW,GW,RW,

TW

(a)ZorbaxSB-C18

(100

×2.1

mm,3.5µm);

(b)ZorbaxEclipse

XDB-C18

(150

×3.0

mm,3.5µm)

(a)0.1%

AcH

andACN

(gradient);(b)ACN

and50

mM

NH4Ac/HAc(gradient)

20–5

00ng

L−1

[42]

3SAs,TMP,

3FQs,

CAP

Urban

waters

ZorbaxEclipse

XDB-c18

(150

×3.0

mm,

3.5µm)

0.05%

form

icacid

andACN

(gradient)

35–1

00ng

L−1

[43]

ORN,TMP,

2SAs,

13FQs

RW

Acquity

UPLC

BEH

C18

(100

×1

mm,

1.7µm)

0.01%

form

icacid

andACN

(gradient)

QqQ

ESI(+)

10ng

L−1

[44]

3MCs,6FQs+2

QdN

Os,16

SAs,

4TCs

WW

(a)and(d):GenesisC18

(50×2.1

mm,

3µm);(b)and(c):GenesisC18

(150

×2.1

mm,3µm)

(a)ACN,20

mM

NH4Ac,

0.05%

form

icacid

(gradient);(b)and(c)ACN,20

mM

NH4Ac,

0.1%

form

icacid

(gradient);(d)ACN,

20mM

NH4Ac,

0.1%

form

icacid

and

4mM

oxalic

acid

(gradient)

QqQ

ESI(+)

1–8

ngL−1

[46]

1PC,2FQs,6MCs,

1LCs,1SA,1TC

(+oP

s)

STPs

LunaC8(50×2

mm,3µm)

(a)0.1%

form

icacid

andACN;(b)0.05%

TEA

andACN

(gradient)

QqQ

ESI(+),

ESI(–)

0.15–

2.08

ngL−1

[47]

ERY,CAP,

SMX

(+oP

s)SW,GW,DW

RP-18WatersXTerra

(100

×2.1

mm,

3.5µm)

MeO

Hand2

mM

NH4Ac(gradient)

(a)QqQ

;(b)QTOF

ESI(+),

ESI(–)

5–10

ngL−1

[48]

SMX,CAP(+oP

s)SW

Acquity

UPLC

BEH

C18

(100

mm

x1

mm,1.7µm)

MeO

H,0.5%

AcH

and10

mM

TrBA

(gradient)

QqQ

ESI(–)

0.05

ngL−1

20ng

L−1

[49]

TMP,

SMX,AMOX,

CAP,

ERY,MET

(+oP

s)

SW,WW

Acquity

UPLC

BEH

C18

(100

×1

mm,

1.7µm)

MeO

HandAcH

(gradient)

QqQ

ESI(+),

ESI(–)

0.1–

2.5

ngL−1

0.2–87

ngL−1

[50]

3TCs,4SAs,TMP,

2LCs,4MCs,2PCs,

4FQs(+oP

s)

SW

Supelco

Discovery

HSC19

(150

×4.6

mm,

3µm)

0.1%

form

icacid

andACN

(gradient)

QqQ

ESI(+),

ESI(–)

0.5–98

ngL−1

[51]

SMX,NAc-SMX,

TMP,

ERY

(+oP

s)WWE,SW

C18

Luna(250

×2

mm,5µm)

40mM

NH4Ac,

form

icacid

andMeO

H(gradient)

ITESI(+)

10–5

ngL−1

[52]

10FQs,6PCs

GW,SW

KromasilC18

(100

×2.1

mm,5µm)

MeO

H,0.1%

form

icacid

(gradient)

(a)QqQ

;(b)QTOF

ESI(+)

0.4–

4.3

ngL−1

[53]

3FQs,1CE,2PCs,

1SA,1NTI,1TC,

TMP

Sew

ageWater

YMCHydrosphere

C18

(150

×4.6

mm,

5µm)

0.1%

form

icacid

andACN

(gradient)

ITESI(+)

0.01–

0.68

ngL−1

[54]

4FQs,3SAs,TMP

WW

ZorbaxSB-C18

(150

×2.1

mm,5µm)

1mM

NH4Ac,

0.007%

AcH

andACN

(gradient)

QESI(+)

2–90

ngL−1

[55]

3MCs,2SAs,4TCs,

7PCs,TMP,

CAP

Water

(a)MachereyandNagel

LiChrospher

100

RP-8

end-capped

(125

×3

mm,5µm);

(b)and(c)Merck

LiChrospher

100RP-18

end-capped

(125

×3

mm,5µm)

(a)10

mM

oxalic

acid

andACN

(gradient);

(b)and(c)10

mM

NH4AcandACN

(gradient)

QqQ

ESI(+),

ESI(–)

20–5

0ng

L−1

[56]

4TCs,8MCs,3SAs,

6FQs,TMP,

CAP,

MET,

NIF

(+oP

s)

Water

Purospher

StarRP-18endcapped

(125

×2.0

mm,5µm)

(a)Water,A

CNandMeO

H(gradient);(b)0.1%

form

icacid

andACN

(gradient)

QqL

ITESI(+),

ESI(–)

0.02

–24

ngL−1

0.1–74

ngL−1

[58]


Tab

le2

(con

tinued)

Com

pounds

Matrix

LC

MS

Sensitiv

ityReferences

Colum

nMobile

phase

Type

Ionizatio

nLOD

LOQ

TMP,

MET,

ERY

(+oP

s)Hospital

WWEs

Purospher

StarRP-18endcapped

(125

×2.0

mm,5µm)

ACN

and0.1%

form

icacid

(gradient)

QqQ

ESI(+)

3.8–

40ng

L−1

11–112

ngL−1

[59]

2MCs,SMX,TMP,

OFLO

(+oP

s)SW,WWI,

WWE

Purospher

StarRP-18endcapped

(125

×2.0

mm,5µm)

ACN,MeO

Hand5

mM

NH4Ac/AcH

(gradient)

QqQ

ESI(+)

1–43

ngL−1

3–120ng

L−1

[60]

ERY,AZI,SMX,

TMP,

OFLO

(+oP

s)GW,RW,

WWI,WWE

WatersAcquity

C18

(50×2.1mm,1.7µm)

5mM

NH4Ac/AcH

,ACN

andMeO

H(gradient)

QTOF

ESI(+)

10–5

00ng

L−1

[61]

13FQs,32

SAs,

12PCs,19

MCs,

5TCs(+

other

veterinary

drugs)

Urine

WatersAcquity

C18

MS

(50×2.1mm,1.7µm)

Formic

acid

andACN

(gradient)

TOF

ESI(+)

0.2–

45µgL−1

[62]

3MCs,3SAs,

TMP(+oP

s)River

sediment

LiChrospher

RP-18

(125

×3mm,5µm)

20mM

NH4AcandACN

(gradient)

QqQ

ESI(+)

3–20

µgkg

−1[63]

6TCs,5SAs

WWI,WWE

XterraMSC18

(50×2.1mm,2.5µm)

0.1%

form

icacid

andACN

(gradient)

ITESI(+)

0.03

–0.07ng

L−1

[64]

5SAs,TMP,

3MCs,

OME,RAN

Sludge

Kromasil100C18

(250

×4.6mm,5µm)

AcH

andACN

(gradient)

QESI(+)

2–8µgkg

−120

–100

µgkg

−1[66]

4MCs,5SAs,TMP

Sludge

Chrom

olith

Perform

ance

RP-18e

(100

x4.6mm)

10mM

NH4AcandACN

(gradient)

QqQ

ESI(+)

3–41

µgkg

−1[69]

6SAs,2PCs

Sludge

AtlantisC18

LC(150

×2.1mm,3µm)

1%form

icacid

andACN

(gradient)

QqQ

ESI(+)

1–270ng

kg−1

5–590ng

kg−1

[70]

TMP,

SMX,TRI

(+oP

s)Soils,digested

sludge

WatersSunfire

(150

×2.1mm,3.5µm)

10mM

NH4AcandACN

(gradient)

ITESI(+)

3–150ng

L−1

[71]

2TCs,1SA,2MCs

AS

XterraMS-C18

(100

×2.1mm,3.5µm)

80mM

form

icacid

andMeO

H(gradient)

QqQ

ESI(+)

0.4–

5.6µgkg

−11.1–

12.8

µgkg

−1[72]

4TCs,2FQs

Wellwater,

RW,STP

Kromasil100C18

(250

×4.6mm,5µm)

AcH

andACN

(gradient)

QESI(+)

4–6ng

L−1

[73]

5TCs,6SAs

GW,SW

LunaC8(100

×4.6mm,3µm)

10mM

ammonium

form

ate,

0.3–0.5%

form

icacid

andMeO

H(gradient)

QESI(+)

100ng

L−1

[74]

ERY,SMX,TMP,

TRI

(+oP

s,endocrine

disruptors

andPCPs)

Water

Synergi

Max

RP

(250

×4.6mm,4µm)

0.1%

form

icacid

andMeO

H(gradient)

QqQ

ESI(+),

ESI(–)

[75]

5SAs,5TCs,7MCs,

TMP,

CAP,

CAR

(+oP

s)

Water

(a)Genesis(150

×2.1mm);

(b)Apex(150

×2.1

mm)

(a)0.015%

HFBA,0.5

mM

NH4Ac,

MeO

HandACN

(gradient);(b)NH4Ac

andACN

(gradient)

QqQ

ESI(+),

ESI(–)

0.03

–1.4

µgL−1

[76]

TMP,

1SA,FLOR,

OXO

SW

LunaC18

(150

×4.6,

5µm)

0.1%

form

icacid

andMeO

H(gradient)

QqQ

APCI(+)

0.51.0ng

L−1

0.7–

1.8ng

L−1

[77]

AMOX,TMP,

SMX

(+oP

s)SW,GW

MetasilBasic

C18

(150

×2.0mm,3µm)

10mM

ammonium

form

ate/form

icacid

andACN

(gradient)

QESI(+)

14–3

0ng

L−1

[78]

6TCs,6SAs,3MCs

RW,river

sediment

XterraMSC18

(50×2.1mm,2.5µm)

0.1%

form

icacid

andACN

(gradient)

ITESI(+)

0.03

–0.07ng

L−1

[79]

2TCs,3SAs,2FQs,

4MCs,TMP

Urban

WW

BetaB

asic

C18

endcapped

(150

×2mm,3µm)

0.1%

form

icacid,0.1%

TFA

andACN

(gradient)

ITESI(+)

1–46

ngL−1

4–152ng

L−1

[80]

13FQs,2SAs,1NTI,

TMP

MW,SW

Acquity

UPLC

BEH

C18

(100

×2.1mm,1.7µm)

0.01%

form

icacid

andACN

(gradient)

QqQ

ESI(+)

0.2–

7.7ng

L−1

0.8–

25.5

ngL−1

[81]

4SAs,4FQs,4TCs,

CAP

GW,LW

,WWI,WWE

DionexAcclaim

C18

(150

×2.1mm,4.6µm)

(a)0.1%

form

icacid

andACN

(gradient);

(b)water

andACN

(gradient)

QqQ

ESI(+),

ESI(–)

0.8–

104.4ng

L−1

[82]

AcH

,acetic

acid;A

MOX,amox

icillin;A

PCI,atmosph

ericpressure

chem

icalionisatio

n;AS,

agricultu

ralsoils;A

ZI,azith

romycin;C

AP,chloramph

enicol;C

AR,carbado

x;CDW,chlorinated

drinking

water;CEs,ceph

alospo

rines;DW,drinking

water;EFFL,effluentsof

mun

icipal

wastewater

treatm

entplants;ENRO,enroflox

acin;ESI-M

S2,electrospray

ionizatio

n–tand

emmassspectrom

etry;

ERY,

erythrom

ycin;FLOR,florfenicol;FQs,

fluo

roqu

inolon

es;GW,grou

ndwater;HFBA,heptafluorob

utyric

acid;IT,iontrap;LCs,

lincosamides;LW

,lake

water;MCs,

macrolid

es;MET,

metronidazole;MW,mineral

water;NAc-SM

X,N-acetylsulfam

etho

xazole;NHAc4,am

mon

ium

acetate;

NIF,nifuroxazide;NMZs,nitroimidazoles;NTIs,nitroimidazoles;OFLO,ofloxacin;

OME,

omeprazole;ORN,ornidazole;OTC,ox

ytetracycline;

OXO,ox

olinic

acid;PCs,

penicillins;PEN

G,penicillinG;QdN

Os,

quinox

alinediox

ides;QqQ

,triple

quadrupo

le;QqL

IT,hy

brid

triple

quadrupo

le–lineariontrap

massspectrom

eter;Q

TOF,

hybrid

quadrupo

letim

eof

flight;Q

s,qu

inolon

es;R

AN,ranitidine;R

W,river

water;S

As,sulfon

amides;S

MX,sulfametho

xazole;S

TPs,sewage

treatm

entplants;SW

,surfacewater;TCs,

tetracyclin

es;TEA,triethylam

ine;

TFA

,trifluoroacetic

acid;TMP,

trim

etho

prim

;TOF,

timeof

flight;TrBA,tributylam

ine;

TRI,

triclosan;

UPLC,

ultraperform

ance

liquidchromatog

raph

y;TW,tapwater;WW,wastewater;WWE,effluent

WW;WWI,influent

WW


and diaminopyrimidines, in the Seine River inner estuaryusing ultraperformance liquid chromatography (UPLC)coupled to MS2 [44]. In a different work, Oasis HLBcoupled to LC-MS2 was used to evaluate the occurrenceand distribution of nine selected antibiotics from fivedifferent antibacterial families—FQs, SAs, diaminopyrimi-dines, TCs and MCs—in a small Mediterranean stream(Arc River, Southern France) [45], and thirty-one antimi-crobials—MCs, Qs, quinoxaline dioxide, SAs, and TCs—infinal (treated) effluents from eight wastewater treatmentplants in five Canadian cities [46].

Other cartridges that have also been tested for theenrichment of antibiotics from aqueous samples includeOasis MCX [47–50], Strata X [51, 52], LiChrolut EN [47],C18 [53], and mixed-phase C2/ENV

+ [54].Oasis MCX is a strong cation-exchange mixed-mode

polymeric sorbent that is capable of both cation-exchangeand reversed-phase interactions. MCX sorbent is based onHLB copolymer, but the additional presence of sulfonicgroups allows for cation-exchange interactions [50]. It canextract acidic, basic and neutral compounds at low pHvalues. Strata X is styrene-divinylbenzene (SDVB) polymerthat has been surface-modified with N-methyl-2-piperidonemoieties that possess both hydrophobic and hydrophilicproperties [51].

The LiChrolut EN cartridge is an ethylvinylbenzene-divinylbenzene copolymer that can extract polar organiccompounds. At pH 7, this polymeric sorbent can also retainneutral drugs through hydrophobic interactions. Castiglioniet al. [47] developed a multiresidue analytical method tomeasure 30 pharmaceuticals belonging to different thera-peutic families, including three MCs, four LCs, two FQs,one SA, one TC and one β-LC, in urban wastewaters. Dueto the different physicochemical properties of these com-pounds, SPE was carried out using two different cartridges:a) LiChrolut EN at pH 7.0 for the extraction of sevenpharmaceuticals, including three LCs (clarithromycin,erythromycin, spiramycin) and one MC (tylosin); b) OasisMCX for the rest. Extraction was carried out at pH 1.5–2with the second cartridge, in the presence of EDTA toprevent the TCs complexing with Ca2+ and Mg2+ ions andresidual metals during the SPE. Recoveries were higherthan 70% in the Oasis MCX cartridges, except foramoxicillin, ciprofloxacin and oxofloxacin (36%, 32%,31%, respectively), and they were slightly lower whenusing the LiChrolut EN (47–64%). Sample analysisrevealed the presence of nineteen pharmaceuticals in thesewage treatment plant at concentrations ranging from 0.5to 2000 ng L−1, including FQs and SAs.

Pozo et al. [53] have described the application ofreversed-phase C18 cartridges, a nonpolar sorbent, for theonline SPE of 16 antibiotics (ten FQs and six β-LCs) inwater. A notable breakthrough was observed for the FQs

that could be reduced by acidifying the samples at pH 2.5with formic acid. This low pH favored penicillin degrada-tion, and the acid had to be added immediately beforeloading the sample into the SPE-LC system.

Isolute C2/ENV+ solid-phase extraction sorbents are based

on highly crosslinked polystyrene polymers that are speciallyderivatized to obtain a wettable surface. They are speciallysuited to the extraction of analytes that are highly soluble inwater and have been applied [54] to the analysis of FQs, β-LCs (penicillins and cephalosphorins), doxycycline, sulfa-methoxazole, TMP and metronidazole in hospital sewagewater. This sorbent does not show ion exchange properties,but the authors found that the use of triethylamine in MeOH(instead of MeOH alone) as an eluting solvent significantlyimproved recoveries (48–90%), probably due to a reductionin the secondary interactions between the analytes and theresidual silanol groups on the particle surface. The authorsreported the presence of antibiotic concentrations on theorder of 100µg L−1 for some antibiotics in the hospital wastewaters, and found large temporal variations in the analyteconcentrations.

Most of the methods described previously were based onoffline SPE procedures, but online procedures have alsobeen reported for multiclass environmental analysis.Feitosa-Felizzola et al. [45] compared an online SPEcoupled to LC-MS2 with the offline mode for the analysisof several classes of antibiotics (TCs, SAs, FQs and MCs)in urban wastewaters. The application of the online modefacilitates method automation, minimizes sample manipu-lation, and reduces analysis time and organic solventconsumption. However, the sample volume is limited bythe system and the enrichment factors can be lower thanthose obtained with offline SPE.

In some applications, two or more SPE cartridges havebeen used either in tandem [55–57] or in parallel topreconcentrate different groups of analytes [47]. Neverthe-less, in most cases, the use of one cartridge is preferred inan effort to simplify the sample preparation procedure asmuch as possible for the simultaneous preconcentration of awide range of pharmaceuticals from different therapeuticgroups, rather than using more complex procedures thatyield higher recovery rates. In that regard, Gros et al. [58]have evaluated the application of MCX and HLB cartridgesin tandem versus the use of the individual cartridgesseparately for the simultaneous preconcentration of 73pharmaceutical residues, including 25 antibiotics, mainlyfrom the MC, TC, FQ and SA families, in surface andwastewater. The use of two cartridges did not significantlyimprove the recovery rates of the target compounds, andOasis HLB cartridges—with EDTA addition prior toextraction—were selected for sample preconcentrationsince they provided the best overall recoveries (from 50%up to 100%, with some exceptions). The addition of the


chelating agent had more of an effect than adjusting thesample pH to acidic values (2.5–3) in terms of enhancingthe retention of FQs, TCs and MCs in the polymericsorbent, so samples were loaded at their natural pH in thepresence of 0.1% EDTA. The use of an analysis methodbased on LC-MS2 and using a hybrid quadrupole–linear iontrap mass analyzer (QqLIT) allowed high sensitivity,selectivity and reliability of results.

In conclusion, the optimization of multiclass antibioticSPE-based preconcentration methods for the analysis ofenvironmental water samples requires careful selection ofboth the nature of the cartridge and the extractionconditions. Sample pH is an important consideration formaximizing the retention of the antibiotics, especiallythose with a strong pH dependence, and avoidingantibiotic degradation [37, 53]. The coextraction of matrixcomponents such as humic and fulvic acids, whichcomplicate the analysis of the more polar antibiotics thatelute at the beginning of the chromatogram in particular,also depends on the pH of the extracted sample and hasbeen found to decrease at neutral pH values versusextraction in acidic media [59–62]. Nevertheless, it hasbeen shown that the presence of humic acids can improvethe extraction of MC antibiotics from spiked groundwater[63].

Chelating agents (such as EDTA) can be added to thesample prior to analysis to remove residual metals from thematrix or glassware, or those sorbed onto the surface ofthe cartridge sorbent, which can cause low extractionrecoveries of MCs, FQs, and especially TCs [39, 47, 58,64]. Salt addition (e.g., sodium chloride) can also beapplied in order to improve antibiotic extraction efficiencyin hydrophilic–lipophilic balance polymers, particularly forSAs and TMP [57, 65].

b) Sewage sludge and sediments

The presence of antibiotics, and pharmaceuticals ingeneral, in solid environmental samples such as sewagesludge or sediments has been studied to a lesser extent thanin water samples, probably due to their greater complexity.However, the analysis of antibiotics in these matrices isimportant, as it allows the eliminating power of sewagetreatment plants (SPTs) to be controlled and the safety ofthe sewage sludge used as manure to be evaluated, andfinally the incorporation of antibiotics into the food chain tobe avoided (Fig. 3) [66].

The main antibiotic classes that have been measured insolid matrices include SAs, MCs, TCs and FQs [14]. Theconcentrations of antibiotics observed in solid wastesamples seemed to be significantly higher than those inaqueous media, suggesting some form of preconcentrationonto the solid samples. These drugs differ considerably interms of their sorption and fixation in soils due to their

different physicochemical properties, such as theirmolecular structures, sizes, shapes, solubilities andhydrophobicities, and also because the cation exchangeproperties, cation bridging, surface complexation andhydrogen bonding can change from sample to sample[67]. Their antibacterial power usually decreases aftersorption and fixation, but in most cases they still producesome antimicrobial effects, which can influence theselection of antibiotic-resistant bacteria in the terrestrialenvironment. For example, FQ antibacterials such asciprofloxacin and norfloxacin have been shown to besorbed specifically to sewage sludge in waste watertreatment plants, and concentrations in the mg kg−1 rangehave been found in digested sludges [68]. TCs have alsobeen shown to bond strongly to soil organic matter due totheir ability to form complexes with doubly-chargedcations, such as calcium, that occur at high concentra-tions in soil [14].

Antibiotics may occur together with other potentiallyinterfering compounds that will require further separationbefore the analysis. Preparative techniques for soils andsludge generally include multistep procedures that firstinvolve the extraction of the antibiotics from the solidsamples into the liquid phase. This step can be based onmechanical shaking or ultrasound-assisted solvent extrac-tion (USE) [63, 69], using high volumes of differentorganic solvents in most cases. Alternatively, pressurizedliquid extraction (PLE) has also been applied, and itprovides high recoveries for most analytes in a shortertime and with decreased organic solvent consumption [66].These techniques are further combined with SPE for sampleclean-up and preconcentration of the target analytes in orderto avoid matrix effects during chromatographic analysis[69, 71].

One of the major challenges in the analysis of solidenvironmental samples is the preparation of synthetic stand-ards for method characterization and validation. To do this, theanalytes must be incubated for a defined time prior toextraction in order to attain sorption–desorption equilibrium.On the other hand, the microbial and enzymatic activities inthe sediments may transform the analytes and/or influencetheir binding to the solid matrix, affecting the extraction yields[32]. In order to minimize the influences of any biotransfor-mation processes, the samples are usually lyophilized beforeanalysis [66, 70]. Other authors have proposed sterilizing thesediments prior to extraction using γ radiation or autoclavingfor example [63].

Díaz-Cruz et al. [70] have described the simultaneousdetermination of eleven antibiotics, nine SAs and two PCs insludge from infiltration basins. The extraction of antibioticswas performed by PLE, mixing the sieved sludge sampleswith EDTA and Hydromatrix. A further clean-up step wascarried out by SPE with an Oasis HLB cartridge. The


recoveries obtained were lower than 67%, with large RSDsranging from 9% to 43% (at spiking levels of 1µg g−1).

Better recoveries were reported by Barron et al. [71] usinga similar method based on PLE followed by SPE for theanalysis of 27 pharmaceuticals, including four antibiotics(TMP, sulfamethoxazole, sulfamethazine and triclosan) frombiosolid-enriched soils and digested sludge. Differentcommercial SPE cartridges were tested for clean-up,including Waters Oasis HLB, Phenomenex Strata X,Varian Focus, Merck LiChrolut EN and PhenomenexStrata X-CW. Final method recoveries of ≥60% wereobtained with the HLB cartridges in soils and digestedsludge. The sample extracts were analyzed by LC-MS2,and it was found that the ion suppression effects arisingfrom matrix components were more pronounced indigested sludge samples than in soils.

Nieto el al. [66] reported a method for the extraction ofthree MCs, five SAs, ranitidine, omeprazole and TMP insewage sludge using PLE followed by LC-ESI-MS withoutfurther clean-up. Recoveries ranged between 54% and 95%for the analysis of a lyophilized sludge sample spiked at2 mg/kg. The inclusion of an SPE step that used Oasis HLBcartridges after PLE to preconcentrate the extracts beforechromatographic analysis did not improve the antibioticrecoveries, which were lower than 50% with the exceptionsof sulfapyridine and tylosin (>68%). This method wasapplied to the analysis of sludge samples from two STPs,and concentrations of between 1.3 and 4 mg kg−1 ofroxithromycin and tylosin were obtained. Some others werefound in several samples at levels below the limit ofquantification, and could be determined with more sensitivedetectors such as MS–MS.

An alternative extraction technique has been optimizedby Löffler et al. [63] using USE for the determination ofthree MCs, three SAs and TMP in river sediments.Antibiotics were extracted first with MeOH, and then byacetone and ethyl acetate, and the collected supernatantswere rotaevaporated and reconstituted with deep groundwater, which is supposed to be free of organic contami-nants. The extracts were spiked with a constant amount ofhumic acids to favor the extraction of the MC antibiotics,and preconcentrated using a mixture of LiChrolute EN andLiChrolute C18 as sorbent [56]. The SPE extracts wereanalyzed by LC-MS2. The performance of the method atlow spiking levels (3 ng/g) was very poor, except forsulfadiazine. Anyhow, the main disadvantages of USE overPLE for extraction from sludge and soil samples include thehigher organic solvent consumption per sample (180 mL[63], versus ~22 mL for PLE [69]) and the lower samplethroughput (~90 min for USE [63], versus ~20 min for PLE[69]), although USE is a simpler and cheaper approach.

Göbel et al. have published a comparative evaluation ofthe performances of USE and PLE for the extraction of

MCs, SAs and TMP from activated and digested sewagesludge [69]. For the PLE method, a MeOH/H2O mixture(50:50, v/v) was selected as extraction solvent using anextraction temperature of 100°C, higher than that used inother applications [66, 71]. For USE, the samples wereultrasonicated for 5 min with MeOH and acetone, and thecombined extracts were evaporated. The extracts werereconstituted with local groundwater for SPE using OasisHLB cartridges. SA antibiotic retention on the SPEcartridges was not affected by the pH of the loadingsamples, as reported for wastewater samples [37]; however,higher RSDs (up to 33%) were observed when the pH ofthe sample was adjusted to 4.0 prior to SPE, due toincreased clogging of the cartridges, which did not allowthe enrichment of the total sample volume in some cases.The precision ranged between 2% and 8% for PLE andbetween 7% and 20% for USE. The lower precision of USEcould be attributed to the higher amount of matrix extractedwith the solvents used in this method and to the higherautomation of the PLE procedure in comparison to USE.However, USE seemed to be equally or slightly lessefficient for the extraction of MCs and TMP, whilesignificantly lower extraction efficiencies were obtainedfor SAs compared to PLE.

Jacobsen et al. [72] reported the simultaneous extractionof two TCs, two MCs and one SA from agricultural soils.They carried out a PLE extraction of all of the antibiotics,followed by a clean-up and preconcentration step usingSPE and then LC-MS2 determination. Two cartridges intandem were applied for sample clean-up and preconcen-tration: a SAX cartridge was employed to remove nega-tively charged humic material and to reduce matrixinterferences, thus avoiding clogging, contamination andoverloading of the HLB cartridge that retained theantibacterial agents. PLE extractions were performed atroom temperature and 1500 psi to avoid the conversionof TCs into their epi or anhydrous form when heated.The sample, with an average moisture content ofapproximately 5%, was mixed with an equal amountof Otawa sand. The extraction solvent was a 1:1 mixtureof MeOH and 0.2 M citric acid buffer (pH 4.7). Theaddition of the chelating agent favored the extraction ofTCs in particular, as they form strong complexes with di-and trivalent cations in the clay mineral interlayers orwith hydroxy groups at the surface of the soil particles.For samples with low concentrations of antibiotics, threePLE extracts were combined prior to preconcentration toimprove method sensitivity. The recoveries varieddepending on the spiked concentration level and thetype of soil, although the extraction procedure wasoptimized for TCs and so higher recoveries couldprobably be achieved for MCs and SAs under differentconditions.


Food matrices

The most common analyzed food matrices include milk,honey, animal tissues and eggs. Sample preparationinvolves procedures for deproteinization, defatting, andsugar hydrolysis for honey samples [33, 83]. Deproteiniza-tion is usually achieved with organic solvents such asacetonitrile or methanol, and, when necessary, sampleextracts are further defatted with hexane.

“Dilute and shoot” is the simplest sample preparationstrategy, especially when designing multiclass methods,where the selectivity of SPE is somewhat of a disadvantage.Dilution of the extracts can reduce matrix effects to acertain degree, but extensive maintenance of the LC-MSsystem is needed to ensure reproducible chromatogramsand MS sensitivity through column regeneration and MSion-source cleaning and/or the use of a divert valve.

As an example, Chico et al. [84] developed a simplemethod for the analysis of 39 antimicrobials (TCs, Qs, PCs,SAs and MCs) in animal muscle tissues. It consisted of anextraction with MeOH/H2O (70:30, v/v) containing EDTAto improve the extraction of TCs followed by the dilution ofthe extracts before injection into the chromatographicsystem. Matrix-matched standards were used for correctquantification of the samples. The simplicity of the samplepreparation procedure along with the use of UPLC enableda high sample throughput to be realized. The method wassuccessfully applied in an Official Public Health Laboratoryfor the routine analysis of 1012 samples over a six-monthperiod [84]. Moreover, the method was applied in severalinterlaboratory studies with good results.

In another application, Granelli et al. [85] developed amethod for the extraction of a total of 19 antimicrobials,including TCs, SAs, Qs, β-LCs and MCs, from muscle orkidney samples. Extraction was performed with 70%MeOH, and the extracts were then diluted fivefold withwater before LC-MS injection. The method was onlysuitable for screening purposes at the MRLs. Matrix effectswere more relevant in kidney samples than in muscle,especially for TCs and MCs, which were affected by signalsuppression, resulting in poorer precision for these twoantimicrobial classes. This can be attributed to the fact thatduring extraction with 70% MeOH, urine salts arecoextracted with the analytes, causing suppression. More-over, lower recoveries were also observed for TC, MCs andQs in kidney samples (<66%) compared to muscle.

A recent work has described, for the first time, a “dilute-and-shoot” strategy for the simultaneous extraction of awide variety of residues and contaminants (pesticides,mycotoxins, plant toxins and veterinary drugs) fromdifferent food (meat, milk, honey and eggs) and feedmatrices [86]. Several antimicrobial classes were included(SAs, Qs, LCs, MCs, IPhs, TCs and NMZs) in the

analytical methodology. Sample extraction was performedwith H2O/ACN or acetone/1% formic acid, but instead ofdiluting the extracts before analysis by ultraperformanceliquid chromatography with tandem MS (UPLC-MS2),small extract volumes (typically 5µL) were injected tominimize matrix effects. Despite the absence of clean-upsteps and the inherent complexity of the different samplematrices, adequate recoveries were obtained for the major-ity of the analyte/matrix combinations (typical values forantimicrobials were in the range 70–120%). Moreover, theuse of UPLC allows high-speed analysis, since all analyteseluted within nine minutes.

Sample preparation strategies known as “QuEChERS”(quick, easy, cheap, effective, rugged and safe), that werepreviously used for pesticide analysis [87], have recently beenapplied to the analysis of multiclass veterinary drugs infoodstuffs. The conventional QuEChERS strategy is basedon ACN extraction/partitioning of the analytes followed bythe removal of water and proteins by salting out with sodiumchloride and magnesium sulfate. Afterwards, dispersive SPE(d-SPE), which involves the addition of small amounts of abulk sorbent to the extracts, is usually applied [87]. Theapplication of this methodology provides—among otheradvantages—high recovery rates for analytes covering awide polarity range, and allows the use of smaller amountsof organic solvents.

For instance, Aguilera-Luiz et al. described a simple andfast procedure for the extraction of SAs, Qs, MCs and TCsfrom milk samples based on a buffered QuEChERS liquidextraction methodology [88]. The target compounds wereextracted from milk with acidified ACN in the presence ofEDTA to increase the recoveries of MCs and TCs. Afterthis step, water and proteins were removed by adding amixture of magnesium sulfate and sodium acetate followedby centrifugation and filtration of the organic phase; thediluted extracts were analyzed directly without furtherclean-up. No denaturing of proteins was required prior toextraction and fat removal, which was performed in a singlestep, so the proposed method is less time-consuming andeasier to perform than the currently available procedures.Furthermore, extraction times were lower than 10 min persample, with recovery values for the antimicrobials rangingbetween 73% and 108%, and separation of the analytes byUPLC was achieved in less than 10 min, so this methodcould be applied in routine laboratories.

In another work, Stubbings et al. [89] demonstrated theapplicability of a QuEChERS procedure for the analysis ofmulticlass antimicrobials (SAs, Qs, FQs, IPhs and NMZs)in animal tissues. Although the conventional QuEChERSliquid extraction method is performed under neutralextraction conditions, the use of buffered acidic conditionswas selected for this work in order to improve theextraction efficiency of Qs/FQs. Thus, extraction was


performed with ACN containing 1% (v/v) acetic acid in thepresence of anhydrous sodium sulfate, followed by d-SPEwith a Bondesil-NH2 sorbent. An aliquot of the extractswas evaporated to dryness and redissolved in ACN/H2O(90:10, v/v) before LC-MS2 analysis. Determination ofNMZs required additional clean-up after d-SPE with BondElut SCX cartridges. Validation was performed on chickenmuscle samples, using matrix-matched standards because ofthe MS suppression signal observed for many of the targetanalytes. Besides the abovementioned antimicrobial classes,recent progress in this line of research has demonstratedthat the method is also applicable to other antimicrobialclasses, namely MCs and LCs.

Yamada et al. [90] have described a method for thesimultaneous screening of 61 antimicrobials of differentclasses along with other 69 veterinary drugs in bovine,porcine and chicken muscle. The samples were firsthomogenized with sodium sulfate and then extracted withACN/MeOH (95:5, v/v). After centrifugation, the extractswere defatted with n-hexane saturated with ACN, evapo-rated to dryness, and the residue was dissolved in MeOHbefore LC/MS2 analysis. The great majority of the analytesshowed recoveries of between 70 and 110%, and onlyciprofloxacin and norfloxacin among the antimicrobialsyielded recoveries of below 70%. Although no significantmatrix effects were observed, poor precisions (RSDs>20%)were obtained for AMPs and SAs.

Didier et al. have recently developed a generic milksample preparation procedure for the comprehensivescreening of 150 veterinary drugs by UPLC-TOF-MS[91]. Among the antimicrobials, β-LCs, MCs, NMZs, Qs,SAs and TCs along with other veterinary medicinalproducts and metabolites were analyzed by the proposedmethodology. To ensure high sample throughput, thesample treatment was kept as simple as possible. Itconsisted of protein precipitation with a solution of 0.1%formic acid in ACN, followed by ultrafiltration using cut-off membranes of 3 kDa. In this way, it was possible toscreen more than 50 samples per day.

A substantial matrix effect was observed that was verycompound dependent. Thus, Qs and TCs suffer significantsignal enhancement (up to 1300% for enrofloxacin),whereas considerable signal suppression was observed foravermectins. In fact, many accuracy values exceeded the120% level, and in some particular cases they even reached807%. So, from a quantitative point of view, the methodpossesses an important limitation, since matrix effects mustbe controlled to avoid important quantitative errors. Despitethat, the signal enhancement was quite beneficial forscreening purposes, as it allows better detection limits.The method was successfully applied to the screening ofmore than 150 raw milk samples as part of the SwissNational plan for residue monitoring.

Honey is a complex matrix that contains sugars, pig-ments and phenolic compounds that must be removed priorto LC-MS analysis [83]. Finding a generic extractionprocedure for multiclass antibiotic analysis in this type ofmatrix is not an easy task. For instance, acid hydrolysis isusually required to dissociate sugar-bound SAs and TCs. Incontrast, MCs are usually extracted under basic conditionsbecause they are not stable at acid pH, and the addition ofEDTA is required to avoid the complexation of theseantimicrobials with metal ions.

Thus, a rather laborious sample preparation procedureconsisting of four subsequent LLE steps has been describedfor the extraction of 37 multiclass antimicrobials (TCs,MCs, AGs, β-LCs, AMPs and SAs) from honey samples[92]. Despite analyzing the extracts independently orpulling them together (which causes β-LC loss due to thepresence of acid in two of the extracts), the authorsperformed a single LC-MS2 analysis using a stackinginjection method. Thus, 10µL of each of the four honeyextracts were successively injected, giving a final injectionvolume of 40µL. The gradient elution and MS/MSacquisition was started after the injection of the last honeyextract. The extract injection order was a very importantinfluence on the β-LC loss during LC-MS2 acquisition. Upto twelve honey samples can be prepared within five hoursusing the abovementioned sample preparation procedure.

Nevertheless, most of the sample preparation methodsfor multiclass antibiotic analysis in food matrices haveadopted SPE clean-up prior to LC-MS determination, eitherto remove matrix interferences or to concentrate theantimicrobials in order to achieve sub-µg kg−1 levelsensitivities. Typical sorbents for SPE include Oasis HLB(hydrophilic–lipophilic balanced) and Strata X, amongothers. Oasis HLB cartridges have been preferred due totheir good retentions and highly reproducible recoveries ofa wide range of compounds, whether polar or nonpolar (dueto their combined hydrophobic–hydrophilic retention mech-anism). Strata X cartridges, which have a similar function-ality to Oasis HLB cartridges, provide comparable results.

SPE is generally performed in offline mode, but in somecases coupling of online SPE with LC-MS through acolumn-switching valve has been used for semi-automatedsample preparation in order to improve sample throughput.Thus, online SPE provides a solution for high samplethroughput and could be more effective when analyzing alarge number of samples in a limited time. However, somedisadvantages of online SPE clean-up include the gradualdeterioration of reusable cartridges and the risk of samplecross-contamination after analyzing incurred samples.

Among the multiclass methods reported in the literature, aprocedure that involves sample dissolution with EDTA undermildly acidic conditions (pH 4.0) followed by SPE with OasisHLB cartridges has been applied for the simultaneous analysis


of MCs, TCs, Qs and SAs in honey samples [93]. Separationand determination by UPLC-MS2 enabled the analysis of 17compounds in less than 5 min. Mean recoveries ranged from70 to 120%, except for three compounds (doxycycline,erythromycin and tilmicosin), which had recoveries of>50%. The method has been applied to the analysis ofhoney samples obtained from different beekeepers and localsupermarkets, and it found residues of erythromycin, sara-floxacin and tylosin in three of them.

Other authors have proposed a sample preparationprocedure that involves the dissolution of honey withwater, centrifugation and filtration, followed by SPE withStrata X cartridges [94]. In this way, 15 antimicrobials fromseven different classes, namely TCs, FQs, MCs, LCs, SAs,AGs and CAP, could be quantified at very low ng mL−1

levels, whereas erythromycin and monensin were onlydetected and confirmed—not quantified—due to the verylow recoveries (~30%) obtained for these two analytes. Themethod has several drawbacks: the analysis of AGs (e.g.,streptomycin) has to be performed separately due to theparticular extraction and chromatographic conditions re-quired, such as the use of ion-pairing reagents; CAP isassayed in a second chromatographic run because thisantibiotic is detected in ESI negative ion mode. Obviously,sample processing and analysis then becomes very timeconsuming, resulting in a low sample throughput.

In another work, Turnipseed et al. described a methodfor the multiclass residue determination of β-LCs, SAs,TCs FQs and MCs in milk and dairy products [95]. Thesample preparation combines extraction with ACN, clean-up with Oasis HLB cartridges, and ultrafiltration usingmolecular weight cut-off filters to improve the overallperformance of the analysis. Acceptable recoveries wereobtained for SAs, MCs and Qs (>70%); however, they wererather low for TCs (50–60%) and β-LCs (<50%). Despitethe extensive clean-up procedure, a large degree of matrixion suppression was observed for many compounds,making it necessary to include matrix-matched calibrationstandards for quantification purposes.

Stolker et al. [96] developed a method that was usefulfor screening more than 100 veterinary drugs in milk,including antibacterials of different classes, namely MCs,PCs, Qs, SAs, TCs, NMZs, IPhs and AMPs. After proteinprecipitation with ACN, centrifugation and further clean-upwith Strata X cartridges, the extracts were analyzed byUPLC-TOF-MS. The results obtained were satisfactory interms of repeatability (RSDs<20% for 86% of thecompounds), reproducibility (RSDs<40% for 96% ofdthe compounds) a,nd accuracy (80–120% for 88% of thecompounds). However, identification criteria for TOF-MSdetectors are not yet included in the EU 2002/657/ECguidelines [97], so the method can only be used forscreening purposes, and those samples that are suspected

to be positive must be confirmed by a tandem MStechnique.

In the same way, Kaufmann et al. have reported amethod based on UPLC-TOF-MS for the analysis of morethan 100 veterinary drugs, including the most relevantantibiotic classes, in different animal tissues (muscle,kidney and liver) [98]. The sample preparation proposedby these authors is rather extensive. It consisted of a liquid–liquid–solid extraction technique (so-called bipolarity ex-traction) which enables polar, medium polar and apolarcompounds to be recovered, followed by SPE clean-up withOasis HLB cartridges. The use of aqueous solvents failed toextract the most apolar compounds, whereas organicsolvents showed poor recoveries for polar PCs and TCs.Thus, an extraction combining two solvents of differentpolarities, such as ACN and aqueous McIlvaine buffer(0.1 M citric acid, 0.2 M disodium phosphate), wasperformed in the presence of high amounts of ammoniumsulfate, which induces phase separation. The formation ofan emulsion favors the simultaneous extraction of a widerrange of analytes in terms of polarity than the use of ACNdissolved in aqueous buffer. Subsequent extract clean-upusing Oasis HLB cartridges allowed the removal of majormatrix food components.

Despite the fact that the sample preparation approachwas rather laborious, recoveries of over 74% were obtainedfor the most of the analytes. Nevertheless, β-LCs wererecovered at lower rates from liver and kidney than frommuscle, probably due to the high enzymatic activity in thesematrices, which is partly responsible for such losses.

A high-throughput method that combines online extrac-tion and determination by LC-MS2 has been developed forthe screening of 13 multiclass antibacterials (MCs, FQs,LCs, and TMP) in different animal muscle tissues [99].After sample deproteinization with ACN, the sampleextracts were directly loaded onto the SPE cartridge,packed with an Oasis HLB sorbent, and connected througha switching valve device to a short LC analytical column.In this way, a complete cycle of SPE clean-up and LCdetermination can be performed in only 6 min. The methodhas shown excellent selectivity, as no interfering peakswere observed in the retention windows of any of the targetcompounds. Furthermore, the performance of the extractioncartridge was found remain consistent over 100 injections.The only precaution taken was to flush ACN and MeOHover the cartridge once the clean-up step was finished, inorder to remove residual tissue matrix.

Heller et al. developed a method to screen antibacterialresidues in table eggs [100]. A total of 29 analytesbelonging to four antibacterial classes (SAs, TCs, FQs, β-LCs) were analyzed. The extraction of the antimicrobialsfrom the matrix was achieved by adding succinate bufferand centrifugation; afterwards, the cloudy extracts required


a clean-up step with Oasis HLB cartridges. Recoveries foreach drug class were as follows: SAs (70–80%), TCs (45–55%), FQs (70–80%), and β-LCs (25–50%). Indeed, thereproducibility of the method varied widely (from 10% to>30%). Therefore, the results provide an estimated con-centration range and the method could be useful forscreening purposes, but not for quantitation.

Pressurized liquid extraction (PLE) possesses greatpotential as a sample preparation technique in foodanalysis, since it combines the benefits of high throughput,automation and low solvent consumption, although expen-sive lab equipment is required [101, 102].

Recently, Carretero et al. described a multiclass methodfor the analysis of 31 antibacterials (including β-LCs, MCs,LCs, Qs, SAs, TCs, NMZs and TMP) in meat samples byPLE-LC-MS2 [103]. Meat samples were homogenized andblended with EDTA-washed sand, and then extracted withwater by applying 1500 psi, 70°C and one extraction cycle(10 min). Besides the automation of the extractionprocedure, a drawback of the method is the large volumesof extract (40 mL) obtained, which required evaporation toreduce the extract volume to 10 mL and thus increasesensitivity. This evaporation step considerably increases thetime required for sample preparation. The proposedmethodology has been applied to the analysis of 152samples of cattle and pig tissues, fand the presence of Qs,TCs and SAs was found in 15% of the samples, although atconcentrations below the MRLs.

Tables 3 and 4 summarize the sample preparationprocedures as well as the analytical methods used todetermine multiclass antibiotics in foodstuff samples.

LC-MS analysis

The majority of the LC methods used for multiclassantimicrobial analysis in environmental and food matrices(Tables 2 and 4) make use of reversed-phase chromatogra-phy with stationary phases based on octadecyl (C18) oroctyl (C8) silane. Some compounds (e.g., AGs), due to theirhighly polar nature, require the use of hydrophobic ion-pairreagents to improve the peak shape and retention inreversed-phase chromatography. It is known that ion-pairreagents can cause ion suppression and produce significantanalyte signal loss, decreasing sensitivity for many com-pounds. For these reasons, it is usually difficult to separateout AGs using current multiclass methods.

Recently, the introduction of UPLC, a fast chromato-graphic technique based on the use of stationary phasescontaining small particles (typically <2µm in size), hasenabled multiclass separations to be performed with higherresolutions, sensitivities and reduced analysis times (aroundthree times shorter than classical LC separation). Thecurrent trend towards more generic and less selective

sample preparation procedures for multiclass analysisincreases the risk of spectral interferences and ion suppres-sion. The use of UPLC could help to reduce matrix effectsproduced by isobaric co-eluting sample compounds be-cause of the enhanced chromatographic resolving powerprovided by UPLC in comparison to conventional LC.Although the application of UPLC is still scarce (Tables 2and 4), it is growing in popularity.

In the 1990s, LC-MS2 operated with electrosprayionization (ESI) or atmospheric pressure chemical ioniza-tion (APCI) sources became mandatory as confirmatorytechniques for the analysis of target veterinary drugresidues, especially in the field of food safety [104].Nowadays, besides conventional tandem mass analyzers,time of flight mass spectrometry (TOF) is emerging as apowerful and complementary technique for the identifica-tion of non-target contaminants (e.g., unknown degradationproducts and metabolites) [105].

Among the tandem mass analyzers applied to multiclassantimicrobial analysis (Tables 2 and and 4), the triplequadrupole (QqQ) is the one that is the most widely usedfor quantitative analyses of target compounds due to itshigh specificity and sensitivity, especially when operated inthe multiple reaction monitoring (MRM) mode [106].According to EU criteria (2002/657/EC) [97] for confirm-ing veterinary drug residues in foodstuffs, at least twoMRM transitions (in the correct ion ratios) must berecorded, usually corresponding to those from the mostabundant precursors to the most abundant product ions.

The major limitation of QqQ working in the MRM modeis that a complete mass spectrum cannot be obtained, sounknown or non-target compounds are missed, thus makingit difficult to detect the presence of novel residues,metabolites or transformation products. Furthermore, de-spite the technological advances in the latest QqQ ana-lyzers, they are limited in terms of the number of MRMtransitions that can be monitored, and thus in their ability todetermine a large number of analytes (over 100) in a singleLC-MS2 run.

On the other hand, the ion-trap (IT) analyzerprovides high sensitivity in full-scan mode and has theability to perform multiple-stage fragmentations (MSn),which is important for the structural elucidation ofunknown metabolites or degradation products, but itssensitivity is one order of magnitude lower than that ofQqQ instruments.

Nowadays, in the field of multiresidue analysis, there isa clear trend towards the use of accurate mass full-scan MStechniques (TOF) [107, 108]. Full-scan MS approachesoffer the ability to screen a virtually unlimited number ofanalytes, including both target and non-target compounds.In the latter case, the possibility of retrospectively evaluat-ing the MS spectra and the isotopic patterns is a useful tool


Tab

le3

Sum

maryof

samplepreparationprocedures

forthedeterm

inationof

multi-classantib

ioticsin

food

samples

Com

pounds

Matrix

Extractionandclean-up

Recoveries(%

)References

SAs,Qs,MCs,TCs,PCs,

TMP

Muscle

Extractionwith

MeO

H/H

2O

(70:30,v/v)

containing

EDTA

,follo

wed

bydilutio

nwith

H2O

andfiltration

60.5–9

6.5%

[84]

SAs,Qs,MCs,TCs,

β-LCs

Muscle,

kidney

Extractionwith

70%

MeO

Handfivefold

dilutio

nwith

H2O

47–1

21%

(muscle);26

–107%

(kidney)

[85]

SAs,Qs,LCs,MCs,

NMZs,IPhs,TCs

Honey,milk

,eggs,

muscle

Extractionwith

H2O/ACN

oracetone/1%

form

icacid

(v/v)

70–1

20%

(>80%

ofanalytes)

[86]

SAs,Qs,MCs,TCs

Milk

“QuE

ChE

RS”liq

uidextractio

nwith

acidifiedACN-EDTA

inthepresence

ofmagnesium

sulfateandsodium

acetate

forremoval

ofwater

andproteins.Centrifugationandfiltrationof

theorganicphaseanddilutio

nof

extracts

73–1

08%

[88]

SAs,Qs,IPhs,NMZs

Chicken

muscle

“QuE

ChE

RS”liq

uidextractio

nwith

1%(v/v)acetic

acid/ACN

andsodium

sulfate,

follo

wed

byd-SPEclean-up

with

aBondesil-NH2sorbent(plusadditio

nalstrong

catio

nexchange

clean-up

with

BondElutSCX

cartridges

forNMZs)

55–8

9%[89]

SAs,Qs,MCs,AMPs,

LCs,IPhs

Muscle

Extractionwith

ACN/M

eOH

(95:5,

v/v)

anddefatting

with

n-hexane

saturatedin

ACN,follo

wed

byevaporationand

dissolutionin

MeO

H70

–110%

[90]

βLCs,MCs,NMZs,Qs,

SAs,TCs

Milk

Deproteinizationwith

asolutio

nof

0.1%

(v/v)form

icacid

inACN,follo

wed

byultrafiltratio

nfor1hwith

cut-off

mem

branes

of3kD

a.Finally,theextractswerepartially

evaporated

andcentrifugedandthesupernatants

collected

Onlyuseful

forscreening;

accuracy

and

precisiondo

notfulfill

EU

guidelines

[91]

SAs,MCs,TCs,β-LCs,

AGs,AMPs

Honey

FoursequentialL-L

extractio

nsteps:(1)ACN;(2)10%

(v/v)TCA

+ACN;(3)NFPA

+ACN;(4)hydrolysis+ACN.

The

four

extractswereindividually

re-suspended

inMeO

H/H

2O

(20/80,v/v),vortexed,sonicated,

andfiltered

TCsandAMPs(68–118%

);AGs,MCs,SAs

andβ-LCs(26–104%

)[92]

MCs,TCs,Qs,SAs

Honey

Extractionwith

aqueousEDTA

undermilk

acidic

conditions(pH

4.0)

follo

wed

byclean-up

with

OasisHLB

cartridges

70–1

20%,except

DOXI,ERYA

andTIL

(>50%)

[93]

TCs,FQs,MCs,LCs,

SAs,AGs,CAP

Honey

Dissolutio

nof

honeywith

water,centrifugatio

nandfiltration.

Analiquotof

thesupernatantwas

dilutedwith

water

forAG

determ

ination.

The

remaining

portionwas

subm

itted

toaclean-up

with

StrataX

cartridges

65–1

04%,except

ERYA

(~30%)

[94]

βLCs,SAs,TCs,FQs,

MCs

Milk

Extractionwith

ACN/0.1%

form

icacid

(v/v)andclean-up

with

OasisHLBcartridges.Partialsolventevaporationfor

20min,follo

wed

byultrafiltratio

nforanother20

min

with

cut-offmem

branes

SAs,MCsandQs(>70%);TCs(50–

60%)

andβ-LCs(<50%)

[95]

MCs,PCs,Qs,SAs,TCs,

NMZs,IPhs,AMPs

Milk

Deproteinizationwith

ACN

(shaking

for30

min)follo

wed

bycentrifugatio

nforafurther15

min.Tenfold

dilutio

nof

thesupernatants

with

H2O

andclean-up

with

StrataX

cartridges

80–1

20%

(for

88%

ofanalytes)

[96]

SAs,Qs,MCs,TCs,

LCs,β-LCs

Muscle,

kidney,

liver

“Bipolarity

”extractio

nwith

ACN-aqueous

McIlvaine

buffer/ammonium

sulfate,

follo

wed

byclean-up

with

Oasis

HLBcartridges

48–1

48%;lower

recovery

ratesforsomeβ-

LCsin

kidney

andliv

erthan

inmuscle

[98]

MCs,FQs,LCs,TMP

Muscle

Hom

ogenization,

deproteinizatio

nwith

ACN,follo

wed

bycentrifugatio

n,partialevaporationof

theextracts,and

defatting

with

n-hexane.After

centrifugatio

nandfiltration,

theextractsunderw

entclean-up

with

anOasisHLB

shortcolumncoupledonlin

eto

LC

>80%

[99]

SAs,TCs,FQs,β-LCs

Eggs

Extractionwith

sodium

succinatebuffer,centrifugatio

n,andclean-up

with

OasisHLBcartridges

45–8

0%,except

β-LCs(<25%)

[100

]

β-LCs,MCs,LCs,Qs,

SAs,TCs,NMZs,TMP

Muscle

PLEextractio

n:solventH2O,pressure

1500

psi,temperature

70°C

,flushvolume60%,static

time10

min,1

extractio

ncycle

70–9

9%[103

]

AGs,am

inog

lycosides;AMPs,am

phenicols;β-LCs,β-lactams;d-SP

E,dispersive

SPE;DOXI,do

xiciclin;ERYA,erythrom

ycin

A;FQs,fluo

roqu

inolon

es;IPhs,iono

phores;LCs,lin

cosamides;

MCs,

macrolid

es;NMZs,

nitroimidazoles;NFPA

,no

nafluo

ropentanoicacid;PCs,

penicillins;PLE,pressurizedliq

uidextractio

n;Qs,

quinolon

es;SA

s,sulfon

amides;TCs,

tetracyclin

es;TIL,

tilmicosin;TCA,trichloroacetic

acid;TMP,

trim

etho

prim


Tab

le4

Sum

maryof

multiclass

LC-M

Smetho

dsused

forantib

iotic

residu

edeterm

inationin

food

samples

Com

pounds

Matrix

LC

MS

Sensitiv

ityReferences

Colum

nMobile

phase

TypeIonizatio

nLOD

orCCα

LOQ

orCCβ

SAs,Qs,MCs,TCs,PCs,

TMP

Muscle

Acquity

UPLCC18(100

×2.1

mm,

1.7µm)

0.2%

form

icacid

with

1mM

oxalic

acid/ACN

with

0.1%

form

icacid

(gradient)

QqQ

ESI(+)

4–318µgkg

−17–

337µg

kg−1

[84]

SAs,Qs,MCs,TCs,β-

LCs

Muscle,

kidney

GenesisC18(50×2.1

mm,4µm)

ACN/0.2%

form

icacid

with

0.1

mM

oxalic

acid

(gradient)

QqQ

ESI(+)

2–15

µgkg

−1–

[85]

SAs,Qs,LCs,MCs,

NMZs,IPhs,TCs

Honey,milk

,eggs,muscle

Acquity

UPLCC18(100

×2.1

mm,

1.7µm),Sym

metry

C18

(150

×3

mm,5µm)

1mM

form

atewith

form

icacid/H

2O:M

eOH(5:95v/v)

with

1mM

form

ate/form

icacid

(gradient)

QqQ

ESI(+)/(–)

0.01

–0.05

mg/

kg−1

–[86]

SAs,Qs,MCs,TCs

Milk

Acquity

UPLCC18(100

×2.1

mm,

1.7µm)

MeO

H/0.01%

form

icacid

(gradient)

QqQ

ESI(+)

1–4µgkg

−13–

10µg

kg−1

[88]

SAs,Qs,IPhs,NMZs

Chicken

muscle

Synergi

FusionC18(100

×2

mm,

2.5µm)

0.1%

form

icacid/MeO

H/ACN

(gradient)

QqQ

ESI(+)

0.3–

444µg

kg−1

0.45

–487

µg

kg−1

[89]

SAs,Qs,MCs,AMPs,

LCs,IPhs

Muscle

TSK-gel

C18100

S(150

×2.1

mm)

10mM

NH4Acwith

0.3%

HAc/ACN-M

eOH

(20:80,

v/v)

(gradient)

QqQ

ESI(+)/(−)

0.03

–3

ngg−

10.1–

10ng

g−1

[90]

βLCs,MCs,NMZs,Qs,

SAs,TCs

Milk

Acquity

UPLCC18(100

×2.1

mm,

1.7µm)

0.1%

form

icacid

inH2O/0.1%

form

icacid

inACN

(gradient)

TOF

ESI(+)

0.2–

222

µg

L−1

0.3–

246

µg

L−1

[91]

SAs,MCs,TCs,β-LCs,

AGs,AMPs

Honey

ZorbaxSBC18(50×2.1

mm,

1.8µm)

1mM

NFPA

with

0.5%

form

icacid/ACN–M

eOH

(50:50,v/v)

with

0.5%

form

icacid

(gradient)

QqQ

ESI(+)

29–8

1µgkg

−1–

[92]

MCs,TCs,Qs,SAs

Honey

Acquity

UPLCC18(100

×2.1

mm,

1.7µm)

MeO

H/0.05%

form

icacid

(gradient)

QqQ

ESI(+)

–0.3–

3.3µgkg

−1[93]

TCs,FQs,MCs,LCs,

SAs,AGs,CAP

Honey

Synergi

Polar

C18(50×2.0

mm,

4µm)

0.1%

form

icacid

inACN/0.1%

form

icacid

inH2O

(gradient)

QqQ

ESI(+)/(−)

––

[94]

βLCs,SAs,TCs,FQs,

MCs

Milk

YMC-A

QC18(100

×2

mm,3µm)0.1%

form

icacid/ACN

(gradient)

QqQ

ESI(+)

–0.02

–25

µg

L−1

[95]

MCs,PCs,Qs,SAs,TCs,

NMZs,IPhs,AMPs

Milk

Acquity

UPLCC18(100

×2.1

mm,

1.7µm)

0.1%

form

icacid/H

2O:ACN

(10:90,v/v)

with

0.1%

form

icacid

(gradient)

TOF

ESI(+)

–1.7–

142

µg

L−1

[96]

SAs,Qs,MCs,TCs,LCs,

β-LCs

Muscle,

kidney,liv

erUPLC

HSST3(100

×2.1

mm,

1.8µm)

0.3%

form

icacid

inACN:H

2O

(5:95,

v/v)/0.3%

aqueousform

icacid

(gradient)

TOF

ESI(+)

–0.1–

30µgkg

−1[98]

MCs,FQs,LCs,TMP

Muscle

LunaC18(50×2.1

mm,5µm)

ACN/0.1%

form

icacid

inwater/M

eOH

(gradient)

QqQ

ESI(+)

0.1–

8.4µgkg

−1–

[99]

SAs,TCs,FQs,β-LCs

Eggs

Phenylcartridgecolumn

(50×4

mm,3µm)

0.1%

form

icacid/ACN

(gradient)

ITESI(+)

10–5

0µgkg

−1–

[100

]

β-LCs,MCs,LCs,Qs,

SAs,TCs,NMZs,TMP

Muscle

XTerra

MSC18(100

×2.1

mm,

3.5µm)

10mM

form

icacid

inH2O/10

mM

form

icacid

inMeO

H(gradient)

QqQ

ESI(+)

12–3

08µgkg

−114

–327

µgkg

−1[103

]

AGs,am

inog

lycosides;AMPs,am

phenicols;β-LCs,β-lactams;CCα,d

ecisionLim

it;CCβ,d

etectio

ncapability;

ESI,electrosprayionizatio

n;FQs,fluo

roqu

inolon

es;IPhs,ion

opho

res;IT,ion

trap;

LCs,lin

cosamides;LOD,lim

itof

detection;

LOQ,lim

itof

quantification;

MCs,macrolid

es;NMZs,nitroimidazoles;NFPA

,non

afluorop

entano

icacid;PCs,penicillins;Qs,qu

inolon

es;QqQ

,triple

quadrupo

le;SA

s,sulfon

amides


in addition to accurate mass determination for detecting andidentifying non-target compounds.

Thus, the coupling of UPLC to high-speed TOFanalyzers provides an excellent analytical tool for themulticlass screening of hundreds of different compoundsin complex matrices [104, 107]. This is particularlyinteresting for environmental or food safety laboratoriesthat have to confirm the presence of a large number ofcontaminants or residues in environmental samples or foodcommodities.

The TOF analyzer offers improved selectivity due to itshigh mass accuracy (1–3 ppm error) and medium massresolving power (typically 10,000 FHWM), which allowsthe identification of mass interferences that have the samenominal masses and chromatographic retention times asanalytes. The disadvantage of the TOF analyzer is itsnarrow linear dynamic range, which somewhat limits itsapplicability to quantitative analysis.

On the other hand, the new hybrid quadrupole time-of-flight (QqTOF) spectrometer can be described as a QqQ inwhich the last quadrupole has been replaced with a TOFanalyzer. A QqTOF can be simply operated as a TOF analyzerin full-scan mode, or as a tandem mass spectrometer in theproduct-ion scanmode. This imparts unique features—such asthe acquisition of full MS spectra with high sensitivity, massaccuracy and medium-range high resolution—that favormultiresidue screening and the structural elucidation ofunknown compounds, as well as accurate mass product-ionspectra that allow the unequivocal identification or confirma-tion of compounds of interest.

However, the sensitivities of TOF and/or QqTOFanalyzers are generally 1–2 orders of magnitude lower thanthat of a QqQ operated in the MRM mode, compromisingtheir applicability, especially in environmental fields,considering the very low concentrations of antimicrobials

normally present in these samples. Table 5 summarizes themain characteristics of the different MS analyzers used forLC-MS determinations of multiclass antibiotics.

Unfortunately, despite the potential of accurate-mass LC-MS technologies for multiclass analysis, the EuropeanCommission Decision 2002/657/EC [97] does not describecriteria for confirming veterinary drug residues in food-stuffs using these medium-range, high-resolution massanalyzers. Decision 2002/657/EC does not take intoaccount an important parameter, mass accuracy, for theconfirmation of a chemical contaminant. However, differentauthors have proposed that additional LC-MS criteriashould be implemented in Decision 2002/657/EC [107].Thus, some have suggested that three TOF ions should becollected in order to earn the 4.0 identification points (IPs)usually required for confirmation when using commontandem MS analyzers [107]. By contrast, others haveproposed a criterion based on the use of either absolute orrelative mass errors for IP assignment, rather than resolu-tion power [109]. Thus, for substances with MRLs, at leasttwo ions must be monitored to achieve a minimum of threeIPs for satisfactory confirmation with mass errors ofbetween 2–10 mDa or ppm.

Conclusions

Recent approaches in antimicrobial analysis have focused onthe development of multiclass analytical methods that allowa realistic evaluation of the fates, bioavailabilities andecotoxicological effects of these drugs in the environment,and enable the food supply to be monitored for regulatedveterinary residues and contaminants, in response to thedemands of regulatory agencies, while minimizing the timeand costs involved.

Table 5 Summary of the different MS analyzers used for LC-MS determinations of multiclass antibiotics

MS analyzer Characteristics Detection capabilities

QqQ High specificity and sensitivity in MRM mode, but complete massspectra cannot be obtained. Low mass resolution. Wide lineardynamic range

Outstanding advantages for quantitative determinations of target analytes, butnot appropriate for identifying unknowns or non-target compounds. Limi-tations when determining a large number of analytes (over 100) in a single run

IT High sensitivity in full scan and multistage fragmentation (MSn)capabilities. Low mass resolution. Narrow linear dynamic range

Useful for screening and identifying unknowns or metabolites. Lacks adequatesensitivity to perform quantitative trace analysis, especially of banned drugs

QqLIT Combines the selectivity of a QqQ with the full-scan highsensitivity of an IT. Low mass accuracy. Wider dynamic rangethan IT

Identification and quantification of target compounds. Identification ofunknowns from fragmentation patterns. Ideal for the confirmation andquantification of compounds that exhibit poor fragmentation

TOF Acquisition of full mass spectra. Mass accuracy (1–3 ppm) andhigh mass resolution (10,000 FHWM). Narrow linear dynamicrange. Lower sensitivity (1–2 orders) than QqQ

Identification of unknowns based on accurate mass and isotopic profileevaluation. Possibility of retrospective analysis

QTOF Acquisition of full or MS2 spectra. Mass accuracy (5 ppm) andmedium-range high mass resolution (5000 FHWM). Lowersensitivity (1–2 orders) than QqQ

Unequivocal identification or confirmation of unknowns based on accurate massand isotopic profile evaluation of product-ion spectra. Possibility ofretrospective analysis

IT, ion trap; MRM, multiple reaction monitoring; QqQ, triple quadrupole; QqLIT, quadrupole–linear ion trap; QTOF, quadrupole time of flight;TOF, time of flight


The method of choice for multiclass antimicrobial analysisis liquid chromatography coupled to MS and LC-MS2.However, despite the high sensitivities and selectivities ofthese techniques, especially LC-MS2, the complexity ofmany food and environmental matrices necessitates clean-upsteps or dilution of the extracts prior to the chromatographicanalysis.

Sample preparation procedures for single-class residueanalysis aim to obtain maximum recovery rates of the analytesin a specific matrix. However, the main goal of theoptimization of multiclass methods is the simultaneousextraction of the largest number of analytes, from multipleclasses, using simple methods in order to achieve high samplethroughput. This requires a compromise between the differentsolid phases that provide the best recoveries for each class ofcompounds. However, the wide variations in the physico-chemical properties of the different antibiotic families and thefact that they are usually present at low concentration levels inenvironmental and food matrices considerably complicatesample preparation, and this issue constitutes one of the mainchallenges for future research in this field.

SPE is the preferred extraction technique for sample clean-up and preconcentration in these samples. Although offlinemethods are applied in most cases, recent developmentsinclude the optimization of online procedures that facilitatemethod automation, minimize sample manipulation, andreduce analytical time and organic solvent consumption.

Regarding chromatographic separations, the introductionof UPLC has facilitated multiclass separations with higherresolutions and sensitivities as well as reduced analysistimes. The availability of this technique is still low, but it isa promising method for future developments. Besidesconventional tandem mass analyzers, recent mass analyzerssuch as time-of-flight mass spectrometers (TOF-MS) andnew hybrid quadrupole time-of-flight (QqTOF) spectrom-eters are emerging as powerful and complementary tech-niques for the identification of new contaminants, althoughtheir sensitivities are generally 1–2 orders of magnitudelower than that of a QqQ operated in the MRM mode,meaning that they may not be applicable to some samples.

Our current knowledge of the impact of antimicrobials inenvironmental and food samples focuses mainly onindividual compounds; however, we also need morescientific research aimed at understanding how the presenceof complex antibiotic mixtures affects human health andenvironmental sustainability.

Acknowledgements This work was funded by the Spanish MEC(grant CTQ2006-15610-C02), the Madrid Regional Government (ref.S-0505/AMB/0374), the ESF, the ERDF, and Complutense University(International Cooperation Projects). Sonia Herranz and ErikaRodriguez thank the Madrid Regional Government and the AlbanProgramme of the European Union (No. E06D101058CO), respec-tively, for a doctoral grant.

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