53
2
Advances in the Analysis
of Pharmaceuticals in the
Aquatic Environment
Sandra Pérez and Damià Barceló
2.1 INTRODUCTION
Recently, the focus of environmental analysis has shifted from the classic contami-
nants, such as the persistent organic pollutants, toward the “emerging contaminants”
detected recently in many environmental compartments.
1
Emerging contaminants
aredenedascompoundsthatarenotcurrentlycoveredbyexistingregulations
ofwaterquality,havenotbeenpreviouslystudied,andarethoughttobepotential
threats to environmental ecosystems and human health and safety. In particular,
the compounds that are being addressed include pharmaceuticals, drugs of abuse,
and personal-care products.
2
The high water solubility of these organic compounds
makesthemmobileintheaquaticmedia,hencetheycanpotentiallyinltratethesoil
and then reach groundwater. Eventually, these compounds may nd their way into
the drinking water supplies.
In recent years the increasing use of drugs in farming, aquaculture, and human
health has become a growing public concern because of their potential to cause
undesirableecologicalandhumanhealtheffects.Themainconcernregarding
Contents
2.1 Int roduction 53
2.2 Multiresidue Methods 56
2.3 Determination of Drugs According to Their Class 67
2.3.1 Analgesics and Antiinammatory Drugs 67
2.3.2 Antimicrobials 68

In general the short- and long-term ecotoxicological
effectsofpharmaceuticalsonwildlifehavenotyetbeenstudiedsufciently.
Prescription and over-the-counter drugs have probably been in the environment
foraslongastheyhavebeenused,butonlyrecentlyhaveanalyticalmethodsbeen
developedtodetectpharmaceuticalsattracelevels.
6
Duetothedilutionandpossible
degradation of these substances in the environment, low levels can be expected.
Therefore, an analyte preconcentration procedure is almost always necessary in
order to achieve desired levels of analytical sensitivity, often requiring high enrich-
me
ntfactors,between100and10,000.Suchenrichmentfactorsfordruganalysis
are usually achieved using solid-phase extraction (SPE). Sensitive detection meth-
ods such as gas chromatography-mass spectrometry (GC-MS), GC-tandem mass
spectrometry (GC-MS/MS) or liquid chromatography-mass spectrometry (LC-MS),
and LC-tandem mass spectrometry (LC-MS/MS) are also crucial for the analyti-
cal
determination of drugs in the environment. The main drawback of GC for drug
analysis,however,isthatthistechniqueislimitedtocompoundswithhighvapor
pressure. Since most drugs are polar substances, they need to be derivatized prior to
injection in the GC. For this reason, the combination of atmospheric pressure ion-
iz
ation-MS (API-MS) with separation techniques such as LC or ultra performance
liquid chromatography (UPLC) has become the method of choice in drug analysis.
LCwithasinglequadrupoleMSanalyzeroffersgoodsensitivity,butwhenvery
complex matrices such as raw sewage are investigated, insufcient selectivity often
impairs the unequivocal identication of the analytes. Tandem MS affords superior
performance in terms of sensitivity and selectivity in comparison with single quad-
ru
pole instruments. Liquid chromatographic techniques coupled to tandem MS or

in inefcient elimination in WWTPs. The removal efciencies vary from plant to
plantanddependonthedesignandoperationofthetreatmentsystems.
12,13
Thus, the
majorsourceofpharmaceuticalresiduesdetectableinsurfacewatersaredischarges
from WWTPs. Several studies reported the occurrence of pharmaceuticals at levels
uptotheμg/Lrangeinrivers,streams,lakes,andgroundwater.
1,14
Rese archershaveyettodeterminetheoccurrence,fate,andpossibleeffectsof
themostfrequentlyconsumeddrugsandtheirmainmetabolitesintheaquaticenvi-
ro
nment. Exceptionally high levels of drugs have been reported—for example, the
occurrenceoftheantiasthmadrugsalbutamolinwaterfromthePoRiver.
15
The
researchersconcludedthattheirdatareectedtheillegaluseofsalbutamolbylocal
farmers to promote growth in cattle. The determination of pharmaceuticals and drugs
of abuse in the environment by appl
yingthep
rinciple that what goes in must come
outcanbeahelpfultooltoestimatethedrugconsumptionintheinvestigatedareas.
For example, Italian researchers
measured the levels of benzoylecgonine, the major
urinarymetaboliteofcocaine,inwastewaterfromseveralItaliancities.
16
What they
foundwassurprising:cocaineuseappearedtobefarhigherthanthepublichealth
ofcials previously thought.
This review provides an overview on analytical protocols used in determining
drugs and some of their metabolites in aqueous and solid environmental samples.

 """!

FIGURE 2.1 Pathways of pharmaceuticals and their metabolites in the environment.
© 2008 by Taylor & Francis Group, LLC
56 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems
Technologicalprogressintheeldsofsampleextractionanddetectionbymassspec-
trometry (MS) techniques (hybrid and tandem mass spectrometers) for analyzing
antibiotics, antiinammatory/analgesics, lipid regulating agents, psychiatric drugs,
sedatives, iodated X-ray contrast media, diuretics, drugs of abuse, and some human
metabolitesintheaquaticenvironmentarediscussed.
17–19
The recent trends in mul-
tiresidue methodologies for the determination of the drugs and their human metabo-
li
teswillbereviewedhere.
2.2 MULTIRESIDUE METHODS
Manyanalyticalmethodologiesforthedeterminationofdrugsinwaterbodiesfocused
on selected therapeutic classes. Multiresidue methods, however, are becoming more
widespreadinresponsetotheneedofmonitoringawiderangeofpharmaceuticals
that belong to diverse drug classes in wastewater, surface water, and groundwater.
Thelatterapproachoffersadvantagesintermsofprovidingamorecomprehensive
picture of the occurrence and fate of the contaminants in the environment. In addi
-
ti
on,thesimultaneousdeterminationofalargenumberofanalytesbyasinglemethod
represents a less time-consuming and hence more economical approach as compared
with applying class-specic analytical protocols. The multiresidue methods found
in the literature are diverse, with target analytes being selected commonly on the
basis of their consumption in the country where the study is being conducted, the
rate of metabolism of drugs, the environmental occurrence, and persistence in the

-
ev
er, low corrected recoveries for oxyphenbutazone, phenylbutazone and 4-amino-
© 2008 by Taylor & Francis Group, LLC
Advances in the Analysis of Pharmaceuticals in the Aquatic Environment 57
antipyridine indicated that the determination of these compounds was still rather
semiquantitative.
20
Vanderford et al.
21
developed an analytical method for the determination of 21
pharmaceuticals in water, choosing them based on their occurrence in the environ-
m
e
nt and their dissimilar structural and physicochemical structures. They used also
a one-step extraction method employing SPE with a hydrophilic-lipophilic balance
(HLB). The separation and detection was performed with LC-MS/MS, using ESI in
either positive or negative mode or atmospheric pressure chemical ionization (APCI)
inpositivemode.Theanalyticalmethodprovidedasimpleandsensitivemethodfor
the detection of a wide range of pharmaceuticals with recoveries in deionized water
above 80% for all of the compounds. The authors also studied the effect of sample
preservatives on the recovery of the pharmaceuticals.
21
They compared formalde-
hyde and sulfuric acid, obtaining the best results for the latter, which prevented the
degradationofthetargetcompoundsanddidnotadverselyaffecttheirrecoveries.
Matrixeffectswerealsoexaminedinthisworkshowingthatallcompoundsdetected
with(+)ESIand(–)ESI,excepthydrocodone,showedaconsiderabledegreeofion
-
ization suppression. Hydrocodone, though, showed signal enhancement. In another

the corresponding concentrations of the standards in the solvent. The authors consid
-
eredthatmatrixeffectswereeliminatedwithdilutions1:2and1:4,andthisapproach
wasselectedforthiswork.Fortheextractionofthetargetanalytes,one-stepSPE
testing Oasis HLB, Isolute ENV+ and Isolute C
18
with and without sample acidi-
cationwasoptimized.OasisHLB,withsorbentbasedonahydrophilic-lipophilic
polymer, provided high recoveries for all target compounds at neutral pH. Recoveries
were higher than 60% for both surface and wastewaters, with the exception of sev
-
e
r
al compounds: ranitidine (50%), sotalol (50%), famotidine (50%), and mevastatin
(34%).
© 2008 by Taylor & Francis Group, LLC
58 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems
Miao et al.
24
reportedamethodusingSPE(C
18
)andLC-(-)-ESI-MS/MSforthe
simultaneousdetectionofnineacidicpharmaceuticaldrugs(bezabrate,clobric
acid, diclofenac, fenoprofen, gembrozil, ibuprofen, indomethacin, ketoprofen, and
naproxen) in WWTP efuents. The recoveries ranged from 59% (indomethacin) to
92%(fenoprofen)intheWWTPefuent.Thespecicityofthemethodwaschecked
spiking samples with analytes at concentration of 0.05 μg/L. Two interfering peaks
resulting from endogenous components in the WWTP efuent were detected in
the MRM channels for fenoprofen and indomethacin. Coextractives in the WWTP
yielded fragmentation patterns similar to fenoprofen and indomethacin. However, the

weremostlygreaterthan70%andinstrumentalandmethodlimitsofdetectionin
the order of ng/L.
Vieno et al.
26
developedamethodthatallowedthequanticationofthefourC-
blockers—acebutolol, atenolol, metoprolol and sotalol, carbamazepine—and the three
uoroquinolones antibiotics—ciprooxacin, ooxacin, and noroxacin—in ground
-
water,surfacewaters,andrawandtreatedsewages.Theauthors
26
studied the effect
ofthewashingandoftheelutingsolventandpHontheextractionstepusingasingle
pretreatment (SPE, Oasis HLB). Prior to the elution step, the adsorbent was washed
with2mLof5%ofmethanolin2%aqueousNH
4
OH showing improvements in the
MS detection in terms of decreased ion suppression and thus improved detectability
of the compounds. Methanol was the solvent of choice yielding the highest recoveries
ascomparedwiththoseobtainedwithacetonitrileoracetone.Thestudyoftheinu
-
e
n
ceofthepHofthewaterintheextractionmethodologywasperformedatthree
pH values: 4.0, 7.5, and 10.0. For most of the compounds, the pH did not have a pro
-
nounced effect on the recovery, with the exception of atenolol and sotalol, which were
poorlyrecoveredatlowpH(<10%atpH4.0).ThesampleswereanalyzedwithLC-
MS/MSusingESIinpositivemodeshowingionsuppressionintheESIsource.
26
To

gical metabolites (1,5-dimethyl-1,2-dehydro-3-pyrazolone, 4-(2-methylethyl)-1,5-
dimethyl-1,2-dehydro-3-pyrazole) were studied. To allow for efcient SPE of the two
microbiological metabolites from water on a conventional C
18
sorbent, the authors
27
prepared the water samples by a simple in situ d
erivatization w
ith acetic anhy-
drideinbasicmediainordertodecreasethepolarityandtoincreasethemolecular
weight of these substances by acetylation. Only the two analytes 1,5-dimethyl-1,2-
dehydro-3-pyrazolone and 4-(2-methylethyl)-1,5-dimethyl-1,2-dehydro-3-pyrazole
werederivatizedwhiletheothercompoundswerequantitativelyextractedwithout
chemical transformation. The analytes were then separated by LC-APCI-MS/MS
and quantied by comparison with the internal standard, dihydrocarbamazepine.
27
AlthoughESIledtohigherpeakintensitiesthanAPCI,thelatterinterfacewascho-
s
e
n because it provided a matrix-independent ionization resulting in recoveries of
~100%
(Table 2.1).
Al
thoughtheuseofGC-MSgenerallyrequiresthederivatizationofpolardrugs,
Boydetal.
28
usedthisapproachtoanalyzeacetaminophen,uoxetine,ibuprofen,
naproxen,andclobricacid,ahumanmetaboliteofclobrateandetobrate.Thetar-
g
e

-
ferentSPEprocedures,andthecombinationofGC-MS(afterderivatizationofthe
acidic compounds) and LC-ESI-MS/MS. Different stationary phases, pH (3, 5, and
© 2008 by Taylor & Francis Group, LLC
60 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems
TABLE2.1
Methods for the Analysis of Drugs in Aqueous Environmental Samples
Analytes Matrix
Extraction Procedure (for
SPE: Sorbent, Sample pH;
Elution Solvent(s))
Separation and
Detection Method Recovery [%]
Limit of Detection and
Quantification Ref.
Multiresidue method for
neutral drugs and 2
metabolites
GW
1
,SW
2
,
WW
3
SPE
4
: Isolute C
18
, pH 7–7.5;

SW:1–30 ng/L
WW:3–160 ng/L
[23]
Multiresidue method for
neutral and basic drugs
GW, SW, WW SPE: HLB, pH 10; MeOH
[Optimization of washing and
eluting solvent, and of pH]
LC-(+)ESI-MS/MS GW: 50–119
SW: 22–113
WW: 64–115
IQL
9
: 0.46–10.6 μg/L
MQL
GW:1–10 ng/L,
SW: 1–24 ng/L,
WW: 1.4–29 ng/L
[26]
Multiresidue method for
neutral and acidic drugs
and 5 metabolites
GW, SW, WW SPE: C
18
; MeOH LC-(+)APCI-MS/MS
Interface
optimization (ESI
and APCI)
GW, SW and WW:87–
117 except for

Cl
2
/MeOH
GC-MS 47–88 IDL:0.6–25.8 ng/L [28]
Combined method for 60
drugs and their metabolites
SW SPE: RP C
18
pH 3, PPL
Bond-Elut pH 7, LiChrolute
EN pH 3, Isolut ENV+ pH
5; MeOH, acetonitrile,
water, triethylamine
GC-MS and
LC-(+)ESI-MS/MS
36–151 IDL: 1.8–13 ng/L [29]
Combined method for 11
drugs and 2 metabolites
SW, WW SPE: Strata X, pH 3; MeOH
[Stationary phase
optimization]
LC-(+/–)ESI-IT-MS >60
Except for lofepramine
and mefenaminic acid
MQL: 10–50 ng/L [30]
Combined method for 11
drugs and 2 metabolites
SW, WW SPE: Strata X, pH 3; MeOH,
MeOH (+2% HOAc),
MeOH (+2% NH

Elution Solvent(s))
Separation and
Detection Method Recovery [%]
Limit of Detection
and Quantification Ref.
Combined method for >50
drugs and their metabolites
SW SPE: 1:tandem HLB and
MCX; MeOH (+5% NH
3
)
2:HLB; MeOH
LC-(+)ESI-MS >80 -10 [10]
Combined method for >10
drugs and 3 human
metabolites
SW, Seawater SPE: Oasis HLB; hexane,
EtOAc and MeOH
LC-(+)ESI-MS/MS
and GC-MS
70–100 MQL:0.07–0.69 ng/L [34]
Combined method for 5
neutral ad acidic drugs and
1 metabolite
SW, WW SPE: Oasis HLB; EtOAc/
acetone (1:1)[Optimization
of washing and eluting
solvent]
GC-MS 71–118 MDL 1–10 ng/L [35]
Methodology by therapeutic

LiChrolute C
18
,pH3;
MeOH
Freeze-drying
LC-(+/–)ESI-MS/MS SPE:
58–120
Freeze-drying: 54–102
MQL
SPE:
2–5 ng/L
MQL
Freeze-drying
: 20–50
ng/L
[46]
13
antimicrobials GW SW, WW SPE: Oasis HLB, pH<3;
acetonitrile (+1% NH
3
)
[Optimization of pH, sorbent
and elution solvent]
LC-(+)-ESI-IT-MS GW
:
51–120
SW: 74–127
WW: 82–126
MDL: 0.027–0.19 μg/L
MQL: 0.10–0.65 μg/L

54–102 MQL
GC-MS: 20–250 ng/L
LC/(+)ESI-MS-MS:10 ng/L-
[48]
4 blood lipid regulators GW, SW, WW SPE: C
18
, pH 7 LC-(+/–)ESI-MS/MS SW: 71–86
WW: 61–91
IDL: 0.7–15.4 pg
MDL: 0.1–15.4 ng/L
[49]
Carbamazepine and 5
metabolites
SW, WW SPE: Oasis HLB, pH 7;
MeOH
[Optimization of stationary
phases]
LC-(+)ESI-MS/MS SW
:
96–103
WW: 84–104
IDL: 0.8–4.8 pg [51]
(Continued)
© 2008 by Taylor & Francis Group, LLC
64 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems
2 antitumoral drugs SW, WW SPE: Macroporous
polystyrene divinylbenzene;
MeOH
LC-(+)ESI-MS/MS 75–102 MDL:
GW: 0.02–0.1 ng/L

MQL:SW: 0.5–1 ng/
LWW: 1–2 ng/L
[57]
6 estrogens, 3 conjugated
estrogens, and 3
progestrogens
Water — GC-MS, LC-MS and
LC-MS/MS
[Optimization of the
interface and
ionization mode]
—IDL:
C/MS: 1–20 ng/mL
LC-ESI-MS: 0.1–20 ng/L
LC-ESI-MS/MS: 0.1–10
ng/L
[58]
1 ICM
11
WW SPE: ENV+, pH 2.8; MeOH LC-(+)ESI-MS/MS 75
MDL: 6.7 ng/L
MQL: 20 ng/L
[12]
TABLE 2.1
(Continued)
Analytes Matrix
Extraction Procedure (for
SPE: Sorbent, Sample pH;
Elution Solvent(s))
Separation and

>90 MDL:
Cocaine :0.12 ng/L
Metabolite: 0.06 ng/L
[16]
1 barbiturate GW — LC-DAD GW: >95 — [69]
6 barbiturates GW, SW, WW SPE: Oasis HLB, pH 7;
acetone and EtOAc
GC-MS GW: 67–104
SW: 64–105
WW: 52–105
MDL:
SW: 1–5 ng/L
WW: 10–20 ng/L
[72]
1
GW: Groundwater
2
SW: Surface water
3
WW: Wastewater
4
SPE: Solid phase extraction
5
MQL: Method quantication limits
6
IDL: Instrumental limit of detection
7
DW: Drinking water
8
MDL: Method detection limit

Phenomenex Strata X). The latter two sorbents were identied as being the most
effective,andStrataXwasshownthebetterphaseforextractingthemajorityofthe
selected compounds. Recoveries typically higher than 60%, except for lofepramine
(not recovered) and mefenamic acid (24%), were found.
30
Forsomepharmaceuti-
cals,ionizationsuppressionduetosolventgradientiscriticalandmustbeoptimized
accordingly for individual analytes. Areas of ion suppression by the matrices were
identied by injecting a blank sample matrix (sewage efuent and freshwater) into a
streamofanalytecausinganelevatedbaseline.
31
Only the suppression of N4-acetyl-
sulfamethoxazole by the efuent matrix was a cause of concern. Another method
using IT-MS in CRM mode for the determination of an innovative list of 10 pharma-
ce
uticals (chloropromazine, chloroquine, closantel, uphenazine, miconazole mid-
az
olam, niumic acid, prochlorperazine, triuoperazine, and triuperidol) listed on
the Oslo and Paris Commission for the Protection of the Marine Environment of the
North East Atlantic (OSPAR) as well as for uoxetine in water was developed.
32
The
limited occurrence of these compounds was thus not surprising, as some
of
them
areusedinfairlysmallquantitiesinthecountrystudied.Threeextractionmateri-
al
s, Oasis HLB, the mixed mode Oasis HLB cation-exchange cartridges MCX, and
Phenomenex Strata X, were tested showing recoveries greater than 60% for the third
extraction material for almost all the compounds except for closantel and cloroquine.

the combination of LC-ESI-MS/MS and GC-MS after
derivatization with methylchloromethanoate for the determination of selected phar-
ma
ceuticals, among them analgesics with emphasis on ibuprofen and its metabolites
(hydroxy-ibuprofen and carboxy-ibuprofen),
C-blockers, a
ntidepressants in wastewa-
ter and seawater, was reported.The e
xtraction procedure was performed in 6-mL
glasscartridgeswiththesamepackingmaterialasinOasisHLBcartridges.Limits
ofquanticationfortheentiremethodwereintherangeof0.07to0.69ng/Lfor
GC-MS,andtherecoverieswerebetween70and100%.Aninterestingresultofthis
work
34
wasthequanticationofibuprofenanditstwometabolitesinthetwotypesof
water showing characteristic patterns, with hydroxy-ibuprofen being the major com-
po
nentinsewage,whereascarboxy-ibuprofenwasdominantinseawatersamples.
The determination of neutral (carbamazepine) and acidic pharmaceuticals (ibupro
-
fe
n, naproxen, ketoprofen, diclofenac, and clobric acid) in surface water and waste-
wa
ter was also performed with SPE using Oasis HLB. Samples were analyzed by
GC-MS after derivatization with diazomethane.
35
Theauthorsanalyzedtheextract
fromSPEtwice,rstdirectlyaftertheSPEmethodandthenafterderivatization.
Recoveries for ketoprofen, diclofenac, and carbamazepine were low when methanol
was used as eluting solvent. Therefore, solvent mixtures of ethyl acetate-methanol

Analgesic and antiinammatory drugs are ubiquitous in wastewater efuents of
municipal WWTPs
36
andasaresultarefoundinsurfacewaters.Thisgroupofcom-
pounds is among the major pharmaceutical pollutants in recipient waters at concen-
tr
ations of up to μg/L levels.
14
Forexample,bezabratehasbeenfoundinWWTP
© 2008 by Taylor & Francis Group, LLC
68 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems
efuent and surface water sample at concentrations as high as 4.6 and 3.6 μg/L,
respectively.
36
Most of the members of this group are acidic in nature because they contain
carboxylic moieties and one or two phenolic hydroxyl groups showing pK
a
values
between3.6and4.9.AtneutralpHtheyexistmainlyintheirionizedform;therefore,
thesamplepHhastobeadjustedtoapHbetween2to3inordertoprotonatethe
carboxylicandhydroxylgroupsinordertoachievehighandreproduciblerecover-
ie
s. First works reported the use of GC-MS or GC-MS/MS for the determination
of these compounds in water matrices. Ternes et al.
37
described a methodology for
the determination of some analgesics, antiinammatory drugs, and lipid regulators
andtwometabolitesofibuprofen(hydroxy-andcarboxy-ibuprofen),togetherwith
compounds such as salicylic acid, the main metabolite of acetylsalicylic acid in sew-
age

it
ednumberofotherions.Forneutralcompoundslikefenoprofen,acetaminophen,
propylphenazone, and phenylbutazone the analysis has been carried out in positive
mode,andallprecursorionsweretheresultof[M+H]
+
of the molecule.
Ananalyticalmethodologyforthedeterminationofveacidicpharmaceuti-
cal
s—ibuprofen, naproxen, ketoprofen, diclofenac, and bezabrate—in water with
OasisMCXandLC-ESI-MS/MSinnegativemodewasdeveloped.
39
Absolute and
relative recoveries (relative to the recovery of the surrogate standard) were reported
for groundwater, surface water, and wastewater. The relative recoveries (Table 2.1)
we
re signicantly higher than the absolute recoveries. The analytical procedure gave
good recoveries for ibuprofen, naproxen, ketoprofen, and diclofenac. Nevertheless,
in the WWTP efuent, the relative recovery of ibuprofen was only 57% and 67%
forbezabrate.Lowmethodlimitsofdetectionwerereportedforibuprofen,diclof
-
en
ac,andbezabrate.Theywere1ng/Lingroundandsurfacewatersand5ng/L
in WWTP samples, and 5 and 25 ng/L for naproxen and ketoprofen, respectively
(Table 2.1).
2.3.2 ANTIMICROBIALS
Antimicrobials are widely used in human and veterinary medicine to prevent or treat
bacterial infections. In addition, veterinary applications include use of antimicrobials
© 2008 by Taylor & Francis Group, LLC
Advances in the Analysis of Pharmaceuticals in the Aquatic Environment 69
as feed additives at subtherapeutic doses to improve feed efciency and promote

+H]
+
.Forthesulfonamidesgroupthe
dominant process from the protonated sulfonamide was the cleavage of the sulphur-
nitrogenbondyieldingthestablesulphanilamidemoietydetectedat
m/z 156. Mac-
rolidesantibioticsarebasicandlipophilicmoleculesthatcontainalactonringand
sugars. These compounds underwent mass fragmentation losing two characteristic
sugars (desosamine and cladinose) and water.
Currently, the methodologies developed for antimicrobial determination include
a list of compounds representative of different classes of drugs because all are
expected to have environmental effects. A methodology for 31 antimicrobials from
the macrolide, quinolone, sulfonamide, and tetracycline classes using SPE and LC-
(+)-MS/MS was developed.
44
Quantitative recoveries for all compounds, even for
tetracyclines, were obtained (
Table 2
.1). The authors used for the extraction of tet-
racyclines Na
2
EDTA as a chelating agent to decrease the tendency for those com-
pounds to bind to cations into the matrix. To improve the resolution and peak shape
of the tetracyclines in the chromatographic column, some authors
45
added oxalic
acid to the mobile phase. In this study
44
the authors used oxalic acid and ESI oper-
ated to 380°C because nonvolatile reagent may accumulate in the ESI source, and

70 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems
However, Na
2
EDTA was added in the freeze drying methodology. Method quanti-
cationlimitsusingSPEwereoneorderofmagnitudelowerduetothe1-Lsample
volumeusedforthedeterminationofantimicrobialsinwater.BattandAga
43
devel-
oped an analytical method for the simultaneous determination of 13 antimicrobials
belonging to 5 classes (uoroquinolones, lincosamides, macrolides, sulfonamides,
and tetracyclines) in wastewater, surface water, and groundwater. The authors opti
-
mized SPE methodology and LC-(+)-ESI-IT-MS as a detection method. The com-
pa
rison between different pH value type of cartridges (C
18
andOasisHLB),and
different eluting solvents was performed.Theo
ptimumconditionprovedtobethe
useofsamplesadjustedtopH3,withNa
2
EDTAadded,andextractionusingOasis
HLB .Theyu
sed Na
2
EDTA to efciently extract the macrolides and tetracyclines.
AlthoughthepHadjustmentdidnotaffecttheextractionefciencyofthemajor-
it
y of the compounds, the recovery for uoroquinolones was reduced below 35% at
no pH adjustment. Fluoroquinolones have exhibited acceptable recoveries in both

(Oasis HLB) and LC-(+)ESI-MS/MS showing recoveries above 80% (Table 2.1),
wi
th the exception of trimethoprim, where they ranged between 30 and 47%, prob-
ab
lybecauseoftheuseofnonidealsurrogatestandard(
13
C
6
Simazine).
2.3.3 ANTIEPILEPTICS, BLOOD LIPID REGULATORS, AND PSYCHIATRIC DRUGS
Some of these classes of compounds are neutral pharmaceuticals without any acidic
functionalgroupsandthereforecanbeenrichedatneutralpHonreversephasemate-
ri
als; they can generally be analyzed by GC-MS without derivatization. However,
LC-MS/MS is the method of choice because it has been shown to have better limits of
detection and better selectivity. Ternes et al.
48
compared the determination by GC-MS
© 2008 by Taylor & Francis Group, LLC
Advances in the Analysis of Pharmaceuticals in the Aquatic Environment 71
and LC-ESI-MS/MS of eight neutral drugs—carbamazepine, clobrate, dimethyl-
aminophenazone, diazepam, etobrate, fenobrate, phenazone, and pentoxifylline.
For three of these drugs—carbamezapine, phenazone, and pentoxifylline—the detec-
ti
on limits were improved to 10 ng/L, independent from the water matrix.
For the analysis of brates and statins, LC-MS/MS with ESI interface is pre-
ferred.Witht h
istechniquethesensitivityisapproximatelytenfoldhigherthanin
an APCI source.
33

51
The developed method encompassed an SPE procedure optimizing the stationary
phase(OasisHLB,Supelclean-18andLC-18)andextractingthewatersamplesatpH
7followedbyseparationanddetectionwithLC-(+)ESI-MS/MS.TheOasisHLBwas
nally chosen for SPE because of its superior extraction efciencies showing recov
-
er
ies for all the analytes including carbamazepine and its metabolites exceeding 80%
inbothwatermatrices.Cross-talkamongsomeMRMchannelswasstudied.The
metabolite10,11-dihydro-10,11-epoxycarbamazepinecouldbeobservedinchannels
m/z 253 o
210 and m/z 253 o 180,whichwereusedtomonitor2-hydroxycarba-
mazepine and 3-hydroxycarbamazepine. Therefore, chromatographic separation of
the analytes was critical and was optimized on a
C8 column using a tertiary solvent
system.
51
All analytes and internal standard were resolved chromatographically with
total run of 11 min. Matrix effects were also studied with four kinds of matrices,
HPLCwater,surfacewater,andinuentandefuentfromaWWTP.Ionsuppression
was highest in the inuent. To correct ion suppression, 10,11-dihydrocarbamazepine
wasusedasinternalstandard.
© 2008 by Taylor & Francis Group, LLC
72 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems
2.3.4 ANTITUMORAL DRUGS
Antitumoral drugs have carcinogenic, mutagenic, teratogenic, and fetotoxic prop-
erties. Cyclophosphamide and ifosfamide, both isomeric alkylating N-lost deriva-
t
i
ves, are among the most frequently used antitumorals. Traces of cyclophosphamide

ti
tation in the low µg/L.
2.3.5 CARDIOVASCULAR DRUGS (C-BLOCKERS) AND C
2
-SYMPATHOMIMETICS
Thesecompoundscontainasecondaryaminoethanolstructureaswellasseveral
hydroxygroups.Duetotheirpolaritythesecompoundsareusuallydeterminedwith
LC-MS/MSandasionizationmodeESIinpositivemodeduetotheirbasiccharac
-
ter. The protonated molecule is the selected precursor ion, and the most intense diag-
no
stic ion is m/z
1
16 corresponding to [(N-isopropyl-N-2-hydroxypropylamine)].
Ternes et al
48
reported the determination of several C-blockers and C
2
-sympatho-
mimetics in waters comparing two methods of separation and detection: GC-MS and
LC-MS/MS. For GC-MS, the sample preparation included SPE (C
18
-endcapped), a
two-step derivatization by silylation of the hydroxyl groups and triuoroacetylation
of the secondary amino moieties. For LC-MS/MS, only the extraction of the water
withSPEwasnecessary.Therecoveriesexceeded70%forbothmethodologies;only
atenolol, sotalol, and celiprolol were not detected by GC-MS. The method limits
ofquanticationwerecomparableforthebothtechniques,being5to10ng/Lin
drinking water and surface water and 50 ng/L in wastewater.
48

with GC-IT-MS/MS was essential, because 17B-ethynilestradiolandan unknown
compound exhibited exactly the same retention time.
Another paper compared different mass spectrometric approaches (derivatized
sample with N,O-bistrimethylsilyl-triuoroacetamide and detected with GC/MS as
well as LC/MS and LC-MS/MS without derivatization) for the analysis of estrogens
(both free and conjugated) and progestogens.
58
For LC-MS and LC-MS/MS, dif-
ferent instruments, ionization techniques (ESI and APCI), and ionization modes
(positiveandnegative)wereemployed.AlthoughLC-ESI-MSshowedinstrumen-
ta
ldetectionlimitscomparablewiththoseobtainedwithLC-ESI-MS/MS(0.1-10
ng/mL), LC-ESI-MS/MS was the method of choice based on the selectivity of this
method that provides the feature to avoid false-positive determinations.
2.3.7 X-RAY CONTRAST AGENTS
IodinatedX-raycontrastmedia(ICM),suchasiopromideanddiatrizoate,arewidely
usedinhumanmedicineforimagingoforgansorbloodvesselsduringdiagnos-
ti
ctests.Theyaremetabolicallystableinthehumanbodyandareexcretedalmost
completelywithinaday.AssuchtheyarefrequentlydetectedinWWTPefuents
andsurfacewatersduetotheirpersistenceandhighusage.
59
Monitoring studies of
iopromideanddiatrizoateinmunicipalWWTPsshowednosignicantremovalof
these compounds throughout the plant.
60,61
The various analytical methodologies commonly used for the determination of
ICMinaqueousmatricesaresummarizedinTable2.1.Allbutonemethodforthe
environmentalanalysisofICMdescribedintheliteratureencompassanenrichment
oftheaqueoussamplebymeansofSPE.Hirschetal.

tionlimitswere50ng/L;lowerdetectionlimitscouldbeachievediftriuoroacetic
acid was used in the mobile phase, but other acid could have affected negatively the
separationoftheICM.Incontrast,inanotherstudy
29
the recoveries obtained for
the four ICM—iopamidol, iopromide, iomeprol, and diatrizoate—were low (<50%)
when only LiChrolute EN sorbent was used. This can be attributed to the extremely
high polarity and water solubility of these compounds.
The same group, in another study
65
using ion chromatography with inductively
coupledplasmamassspectrometry(IC-ICP-MS)withoutprevioussamplepre-
co
ncentration,foundthatlimitsofdetectionbelow0.2µgL
-1
couldbeachieved.
Reproducibilitywasbelow6%forthesixICMstudied.Comparingthesensitiv-
it
y and specicity of the two methodologies, direct injection and detection IC-ICP-
MS
65
and SPE and LC-MS/MS,
29
reported by the same group, LC-MS/MS offered
asignicantlyhighersensitivity(MDLbelow10ng/L)andspecicity.However,
theIC-ICP-MSmethodofferedthepossibilityofdetectingotheriodine-containing
compounds besides the target analytes.
ForthedeterminationofICMinenvironmentalsamples,ESIusuallyoperated
in the positive ion mode has been the preferred method for the sensitive detection
ofthesepolaranalyteswithmolecularweightsofupto1600Da.Formonomeric

LC-(+)ESI-MS/MS and LC-ESI-IT-MS. This was presented as a “nonintrusive”
© 2008 by Taylor & Francis Group, LLC
Advances in the Analysis of Pharmaceuticals in the Aquatic Environment 75
approach to determine abuse drug usage in the community. Recoveries were >90%
forbothcompounds,andmethodlimitsofdetectionwere0.06and0.12ng/Lfor
benzoylecgonine and cocaine, respectively.
2.3.9 OTHER DRUGS
Barbituratesarederivativesofbarbituricacidandactascentralnervoussystem
depressants; therefore, they produce a wide spectrum of effects—from mild seda-
ti
on to anesthesia. Some are also used as anticonvulsants. Today, barbiturates are
infrequently used as anticonvulsants and for the induction of anesthesia.
68
Benzodi-
azepines were mainly used as replacements, and since the introduction of diazepam
(the rst benzodiazepine prescribed for clinical use) in 1963, barbiturates have been
graduallyphasedout.Nowadays,duetolowusage,fewreportsofbarbituratesin
the environment are reported. However, two studies recently pointed out the need
to investigate these compounds in the environment. Holm et al.
69
rst reported on
leachatescarryingpharmaceuticalsfromalandll.Highconcentrations(mg/L)of
numerous sulfonamides and barbiturates (5,5-diallylbarituric acid) analyzed with
LC-Diode array detector (DAD) from domestic waste and from a pharmaceutical
manufacturerwerefoundinleachatesclosetothelandll.Twostudiesalsoreported
the occurrence of barbiturates in the environment, pentobarbital and 5,5-diallylbari
-
tu
ricacidingroundwater
70

-
nat
iondependsonthetypeofstationaryphaseandthewashingandelutingsolvents
used for that purpose, as has been described in this chapter. The determination of
human metabolites of drugs is challenging, because these biotransformation prod
-
uctsareusuallymorepolarthantheparentcompoundandtheymightnotberetained
© 2008 by Taylor & Francis Group, LLC
76 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems
by conventional sorbents. Some authors reported the addition of derivatizing agents
to the water in order to allow for efcient extraction of these polar metabolites with
conventional extraction procedures. An additional challenge is the determination of
someexcretedmetabolites.Theyareformedbyconjugationwithglucuronicacid
orotherpolarmoietiesthatareexpectedtobecleavedbymicroorganismsintothe
unchanged pharmaceuticals in the environment.
LC-MS/MSisthemostfrequentlyemployedseparationanddetectiontechnique
in drug analysis, due to its high sensitivity and because it allows for unequivo-
cal
identicationoftheanalytes.AlthoughtheuseofthistechniqueinESImode
exhibits several advantages, ion suppression is an important process to be taken into
account in the quantication of the analytes in view of the reported studies showing
drasticmatrixeffectswhenESIsourcewasused.
Matrix effects (signal suppression or enhancement) are believed to result from the
competition of the analyte ions and the matrix components for access to the droplet
surfacetothegas-phaseemission.Afeasiblesolutiontoaddressthisissueistouse
standard addition, but this is time consuming and cost intensive. Another approach
reliesontheuseofanAPCIsource,whichismuchlesssubjecttomatrix-dependent
ionization interferences. Although for many polar compounds ESI usually leads to
higherpeakintensitiesascomparedwithAPCI,ESIsignalsforthesecompounds
canbeaffectedbythematrixofthesample.Tominimizematrixeffectsanoptimi

4:711.
© 2008 by Taylor & Francis Group, LLC
Advances in the Analysis of Pharmaceuticals in the Aquatic Environment 77
3. Jones, O.A., Lester J.N., and Voulvoulis, N. 2005. Pharmaceuticals: a threat to drinking
water? Trends Biotechnol.
23:163.
4. M
ellon, M., Benbrook, C., and Benbrook, K.L. Hogging it: estimates of antimicrobial
abuse in livestock.ARe
portofUnionofConcernedScientists,Cambridge,MA.2001.
5. Schulman, L.J., Sargent, E.V., Naumann, B.D., Faria, E.C., Dolan, D.G., and Wargo,
J.P. A human health risk assessment of pharmaceuticals in the aquatic environment.
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Hum. Ecol. Risk. Assess. 8
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6. E
rickson, B.E. Analyzing the ignored environmental contaminants. 2002. Environ.
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6:141A.
7. H
opfgartner, G., Varesio, E., Tschäppät, V., Grivet, C., Bourgogne, E., and Leuthold,
L.A.2004.Triplequadrupolelineariontrapmassspectrometerfortheanalysisof
small molecules and macromolecules.
J. Mass Spectrom. 3
9:845.
8. H
irsch, R., Ternes, T., Haberer, K., and Kratz, K.L. 1999. Occurrence of antibiotics in
the aquatic environment. Sci. Total Environ. 2
25:109.
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9:5157.
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erer, T. 2002. Occurrence, fate, and removal of pharmaceutical residues in the
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alsch, W. 1999. Biodegradation of the iodinated X-ray contrast media diatrizoate and
iopromide. Sci. Total Environ. 2
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