Fate of Pharmaceuticals in the Environment and in Water Treatment Systems - Chapter 2 docx

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

2.3.3 Ant iepilept ics, Blood Lipid Regulator s, a nd Psych iatr ic D rugs 70
2.3.4 Antitu moral Dr ugs 72
2.3.5 Cardiovascular Drugs (C-Blockers) and C
2-
Sympathomimetics 72
2.3.6 Estrogens 72
2.3.7 X-Ray Contrast Agents 73
2.3.8 Drugs of Abuse 74
2.3.9 Other Drugs 75
2.4 Conclusion 75
Ack nowledgments 76
References 76
© 2008 by Taylor & Francis Group, LLC
54 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems
pharmaceuticals is that they are being introduced continuously into water bodies
aspollutants,andduetotheirbiologicalactivitythiscanleadtoadverseeffectsin
aquatic ecosystems and potentially impact drinking water supplies.
3
Antibiotics are
oneofthemostproblematicgroupsofpharmaceuticals,sincetheirincreasingusefor
more than four decades has led to the selection of resistant bacteria that can threaten
the effectiveness of antibiotics for the treatment of human infections.
4
Another group
that has caused environmental concern is contraceptives and other endocrine disrup-
to
rs; due to their endocrine properties they can induce the feminization or masculin-
iz
ation in aquatic organisms.
5

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

hybrid mass spectrometers with distinct analyzers such as triple quadrupole (QqQ),
time-of-ight (ToF), quadrupole time-of-ight (QqToF), quadrupole ion trap (IT),
and recently the quadrupole linear ion trap (QqLIT) are the most widely used instru-
me
ntal techniques for drug analysis.
7
Most of the data on the presence on pharmaceuticals in wastewaters, rivers, and
drinking water come primarily from European studies
8,9
followed by those carried
outintheUnitedStates.
10
These substances that are used in human and veterinary
medicinecanentertheenvironmentviaanumberofpathwaysbutmainlyfrom
discharges of wastewater treatment plants (WWTPs) or land application of sew-
age
sludge and animal manure, as depicted in Figure 2.1. Most active ingredients
of pharmaceuticals are transformed only partially in the body and thus are excreted
as a mixture of metabolites and bioactive forms into sewage systems. Therefore, the
treatment of wastewaters in WWTPs plays a crucial role in the elimination of phar-
ma
ceuticalcompoundsbeforetheirdischargeintorivers.Duringtheapplicationof
© 2008 by Taylor & Francis Group, LLC
Advances in the Analysis of Pharmaceuticals in the Aquatic Environment 55
primary and secondary treatments, pharmaceutical compounds can be eliminated
by sorption onto the sludge or microbially degraded to form metabolites that are
usuallymorepolarthantheparentdrugs.
11
Inmanycasesthehighpolaritycombined
with the low biodegradability exhibited by some pharmaceutical compounds results

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

environment. In this chapter, multiresidue methods for drug analysis in the aquatic
environment are reviewed.
Ternes et al.
20
reported the determination of neutral pharmaceuticals: propyphen-
azone (analgesic), phenylbutazone (antiinammatory), diazepam (psychiatric drug),
omeprazole (antiulcer), nifedipine (calcium antagonist), glibenclamide (antidiabetic),
and two human metabolites: 4-aminoantipyridine (metabolite of metamizole) and
oxyphenbutazone (metabolite of phenylbutazone) with a multiresidue methodology
including a one-step extraction method based on SPE with RP-C
18
material eluted
with methanol. The analysis was performed by LC with detection by electrospray
ionization(ESI)tandemMSinmultiplereactionmonitoring(MRM)mode,which
is the acquisition mode providing the best sensitivity and selectivity for quantitative
analysis. Low limits of detection and reasonable recoveries for the selected drugs in
differentmatriceswereachieved.Thiswork
20
investigated the losses in the recover-
iesofsomedrugsduetomatriximpurities,whicheitherreducedthesorptionef-
ci
encies on the C
18
material or led to signal suppression in the ESI interface. The
authors spiked inuent wastewater extracts with the target analytes and found that
the recoveries were not appreciably higher in comparison to the recoveries over the
total method. Consequently, the signal suppression in ESI played a decisive role
in the losses of 4-aminoantipyrine, omeprazole, oxyphenbutazone, phenylbutazone,
and propyphenazone. For most of the compounds, compensation for the losses was
achieved by addition of the surrogate standard 10,11-dihydrocarbamazepine. How

-
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

workthesamegroupreportedamethodologyusinganisotopedilutiontechniquefor
every analyte to compensate for matrix effects in the ESI source, SPE losses, and
instrument variability.
22
The method was tested with three matrices (wastewater,
surface water, and drinking water), and the results indicated that the method was
very robust using isotope dilution for each target compound. The work described a
method that analyzed 15 pharmaceuticals and 4 metabolites using SPE (HLB) cou
-
p
l
ed with LC-MS/MS with ESI source. Matrix spike recoveries for all compounds
were between 88 and 106% for wastewater inuent, 85 and 108% for wastewater
efuent, 72 and 105% for surface water impacted by wastewater, 96 and 113% for
surfacewater,and91and116%fordrinkingwater.Themethoddetectionlimits
werebetween0.25and1ng/L.
Astudy
23
evaluated different strategies to reduce matrix effects in LC-MS/MS
withanESIinterface.First,thepeakareaofthetargetcompoundsinsolventwere
compared with the target compounds spiked in matrix extracts obtaining signal sup
-
pressions in the range of 40 to 90%. Next, internal calibration curves with inter-
n
a
l labeled standard in solvent and in spiked matrix extracts were prepared. The
overlapping of both curves conrmed that signal losses experienced by the analytes
were corrected by the internal standards. Finally, the effectiveness of diluting sample
extracts was studied. For this purpose, the signals obtained after sequential dilution
of a WWTP efuent and inuent extract were compared with the ones obtained for

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

separationefciencyprovidedbyhighperformanceliquidchromatography(HPLC)
was sufcient to resolve these interfering compounds from the analytes showing the
importanceofusingLCtoimprovetheselectivityoftheanalysis.
AmultiresidueanalyticalmethodusingSPEandLC-MS/MSfor28pharma
-
ceuticals including antimicrobial drugs, two diuretics (furosemide and hydrochlo-
r
o
thiazide), cardiovascular (atenolol and enalapril), antiulcer, psychiatric drugs, an
antiinammatory, a
C
2
-sympathomimetic (salbutamol), a lipid regulator, some estro-
gens (17C-
estradiol, 1
7B-ethinylestradiol, estrone), two antitumorals (cyclophospha-
mide and methotrexate), and two metabolites (clobric acid and demethyl diazepam)
was developed.
25
To optimize the extraction method, several stationary phases and
different PH samples were tested. The cartridges were Oasis MCX at pH 1.5/2.0
and3.0forallthecompoundsandatpH7.0/7.5foromeprazole;LiChroluteENat
pH 3.0, 5.0, 7.0, and 9.0 for all the compounds; Bakerbond C
18
at pH 8.0 and 9.5
for the extraction of amoxicillin;a
nd O
asisHLBatpH7.0foromeprazoleandpH
8.5/9.0 for amoxycillin. They selected Oasis MCX for water samples at pH 1.5/2.0
and LiChrolute EN for water samples at pH 7. Recoveries of the pharmaceuticals

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

evaluate the matrix effects, the authors infused continuously a standard solution into
themassspectrometerandtheninjectedeithersolventorarealsampleextractonto
theLCcolumn.Moreover,SPEextractsofgroundwater,surfacewater,andwastewa
-
t
e
rinuentandefuentwerespikedwithpharmaceuticals,andspikedsampleswere
© 2008 by Taylor & Francis Group, LLC
Advances in the Analysis of Pharmaceuticals in the Aquatic Environment 59
analyzedinLC-MS/MSwithESIinterface.Noionsuppressionwasnoticedforany
of the analytes in groundwater extracts; some signal suppression (<8%) was noticed
for sotalol, acebutolol, and metoprolol for the surface-water extract; and more severe
signal suppression (40%) was observed in the wastewater inuents and efuents. The
authors
26
reported relative recoveries higher than those reported previously by other
authors
24
showinganimprovementofthegeneralmethodologyforallthetargetana-
lytes, except for ciprooxacin and noroxacin.
The determination of selected drugs and their metabolites with a multiresidue
methodology was also reported by Zuehlke et al.
27
Carbamazepine, dimethylami-
nophenazone, phenazone, propyphenazone, 1-acetyl-1-methyl-2-dimethyloxanoyl-2-
phenylhydrazide, 1-acetyl-1-methyl-2-phenylhydrazide, two human metabolites of
metamizole (formylaminoantipyridine and aminoantipyridine), and two micobio
-
l
o

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

tcompoundswereisolatedfromwastewater,surfacewater,anduntreateddrink-
i
n
gwatersamplesbySPEusingapolarSDB-XCEmporedisk.Derivatizationwith
N,O -bis(trimethylsilyl)-triuoroacetamideinthepresenceoftrimethylchlorosilane
wasusedtoenhancethermalstabilityofclobricacid,whichthermallydegradedin
theGCinjectionport,andtoreducethepolarityofspecictargetanalytes(clobric
acid,ibuprofen,andnaproxen)inordertofacilitatetheirGC-MSanalysis.Thelimits
ofdetectionwerebetween0.6(clobricacid)and25.8ng/L(uoxetine).Although
therecoveriesformostofthecompoundsweregreaterthan47%(Table2
.1), a
cet-
am
inophen was repeatedly not detected possibly due to the weak retention of this
compound on the extraction disk.
Next, analytical methods using multiple extraction methods, different liq
-
u
i
d chromatography eluents, or the combination of two detection techniques are
reviewed.
10,29,30
Sacher et al.
29
reported the determination of 60 pharmaceuticals
including analgesics, antirheumatics,
C-blockers, b
roncholitics, lipid regulators
including two metabolites, antiepileptics, vasodilators, tranquilizers, antitumoral
drugs, iodated X-ray contrast media and antimicrobials in groundwater with dif

-
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;

MeOH
LC-(+)ESI-MS/MS GW>80
SW and WW: 9–97%
MQL
5
GW:10 ng/L
WW: 25–250 ng/L
[20]
Multiresidue method for
neutral and acidic drugs
SW, WW SPE: HLB, pH 2; MeOH LC-(+/–)ESI-MS/MS
Deionized water: 80 IDL
6
:12–32 ng/L [21]
Multiresidue method for 15
drugs and 4 human
metabolites
DW7, SW, WW SPE: HLB; MeOH/MTBE
(1:9)
LC-(+/–)ESI-MS/MS 90–110 MDL: 0.25–1 ng/L [22]
Multiresidue method for
neutral and acidic drugs
and 5 metabolites
SW, WW SPE: HLB, pH 7; MeOH
[Optimization of stationary
phases and pH]
LC-(+/–)ESI-MS/MS SW: 50–116
WW: 60–102
MDL
8

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

dimethylaminophenazone
MQL: 10–20 ng/L [27]
Multiresidue method for
acidic drugs
WW SPE: LiChrolute 100 RP-18,
pH 2; MeOH
LC-(–)ESI-MS/MS 59–92 MDL: 5–20 ng/L [24]
© 2008 by Taylor & Francis Group, LLC
Advances in the Analysis of Pharmaceuticals in the Aquatic Environment 61
Multiresidue method for 28
drugs and 2 metabolites
WW SPE: 1 Oasis MCX, pH
1.5/2.0; MeOH, MeOH
(+2% NH
3
) and MeOH
(+0.2% NaOH)
2. LiChrolute EN, pH 7;
MeOH, EtOAc
[Optimization of stationary
phases and pH]
LC-(+/–)ESI-MS/MS >70 IQL: 600–39400 ng/L
MQL:0.1–5.2 ng/L
[25]
Multiresidue method for 4
drugs and 1 human
metabolite
DW, SW, WW SPE: SDB-XC, pH 2; MeOH,
CH
2

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

3
)
[Stationary phase
optimization]
LC-(+/–)ESI-IT-MS >60
Except for chloroquine
and chlosantel
MDL: 1–20 ng/L
IQL: 20–105 pg
[32]
Combined method for 13
drugs
SW, GW, DW SPE: Oasis-MCX, pH 3;
MeOH/NH
3
(19:1)
LC-(+/–)ESI-MS/
MS and
LC-QqToF-MS
60–75 except for
fenobrate (36)
MQL: 5–25 ng/L [33]
(Continued)
© 2008 by Taylor & Francis Group, LLC
62 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems
TABLE 2.1
(Continued)
Analytes Matrix
Extraction Procedure (for
SPE: Sorbent, Sample pH;

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

class
Acidic drugs, acetylsalicylic
acid and 4 metabolites
SW, DW, WW SPE: C18, pH 2; MeOH GC-MS and
GC-IT-MS/MS
58–90 MQL:
WW: 50–250 (GC-MS)
SW:5–20 (GC-MS)
DW: 1–10
(GC-IT-MS/MS)
[36]
8 acidic drugs SW SPE: C18, pH 2; MeOH GC-IT-MS/MS ≤90
MQL:1 ng/L [38]
5 acidic drugs GW, SW, WW SPE: Oasis MCX, pH 2;
acetone
LC-(–)ESI-MS/MS GW:
82–103
SW: 75–112
WW: 57–100
MQL:1–25 ng/L
[39]
© 2008 by Taylor & Francis Group, LLC
Advances in the Analysis of Pharmaceuticals in the Aquatic Environment 63
31 antimicrobials WW SPE: (1) Oasis HLB, pH 6
(2) Oasis HLB, pH 3 and
Na
2
EDTA; MeOH
LC-(+)ESI-MS/MS 72–99 MDL: 1–8 ng/L [44]
18 antimicrobials GW, SW SPE: LiChrolute EN +

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

[43]
13 antimicrobials SW SPE: Oasis HLB, pH 2–3;
MeOH
LC-(+)-ESI-IT-MS
and LC-DAD
96–102 MDL:
0.05 μg/L
MDL
US EPA
: 0.03–0.5 μg/L
MQL: 0.1 μg/L
[47]
13
antimicrobials and 1
human metabolite
WW SPE: Oasis HLB, pH 4;
MeOH/EtOAc (1:1) and
MeOH (+1% NH
3
)
LC-(+)ESI-MS/MS 89–108 (trimethoprim:
47)
MQL:
0.3–77 ng/L
[42]
8
neutral drugs DW, SW, WW SPE: C
18
, pH 7.5; MeOH GC-MS and
LC/(+)ESI-MS/MS

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

SW: 0.02–0.1 ng/L
GW: 0.3–2 ng/L
[54]
3 antitumoral drugs WW (Hospital
efuents)
SPE: C
8
, pH 7.5; MeOH/
CHCl
3
(1:2)
LC-Fluorescence 85–87 MDL: 0.05–0.06 μg/L
MQL: 0.26–0.29 μg/L
[55]
C-#lockers and
C
2
-symphatomimetics
SW, GW, WW SPE: C
18
-endcapped, pH 7.5;
MeOH
GC-MS and
LC-(+)ESI-MS/MS
>70 MQL:
SW and GW: 5–10 ng/L
WW: 50 ng/L
[48]
7 estrogens SW, WW SPE: acetone GC-IT-MS/MS SW: 41–90
WW: 56–82

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

Detection Method Recovery [%]
Limit of Detection
and Quantification Ref.
© 2008 by Taylor & Francis Group, LLC
Advances in the Analysis of Pharmaceuticals in the Aquatic Environment 65
6 ICM DW, SW, WW SPE: ENV+, pH 2.8; MeOH
[Optimization of stationary
phases]
LC-(+)ESI-MS/MS SW: 90–116
WW: 57–113
(ioxithalamic acid: 35)
MQL:10–50 ng/L [62]
4 ICM and 2 possible
human metabolites of ICM
SW, WW SPE: ENV+ and Envi-Carb,
pH 3.5; MeOH and
acetonitrile/water (1:1) back
ush
LC-(+)ESI/MS-MS 55–100 MDL: 50 ng/L [63,
64]
4 ICM GW SPE: ENV, pH 3; MeOH LC-(+)ESI-MS/MS 9–46
MDL: 2.3–4.8 ng/L [29]
7 ICM SW, DW Direct injection IC-ICP-MS 97–99 IDL: 0.02–0.04 µg/L [65]
Cocaine and its metabolite
(abuse drug)
SW, WW SPE: Oasis HLB, pH 2;
MeOH and MeOH (+2%
NH3)
LC-(+)ESI-MS/MS
and LC-ESI-IT-MS

>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

9
IQL: Instrumental quantication limit
10
Not reported
11
ICM: Iodinated Contrast Media for X-ray
© 2008 by Taylor & Francis Group, LLC
66 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems
7), and eluting solvents were used for the determination of the target compounds.
Recoveriesvariedbetween75and125%inbothtapwaterandsurfacewater,and
limitsofdetectionwereverylowforsomeofthetargetcompounds(Table 2.1).
Ac
ombined method was developed for the determination of 11 pharmaceuticals
(dextropropoxyphene, diclofenac, erythromycin, ibuprofen, lofepramine, mefenamic
acid, paracetamol, propanolol, sulfamethoxazole, tamoxifen, trimethoprim) and 2
metabolites (N4-acetyl-sulfamethoxazole and clobric acid). The method relied on a
one-step-SPE, an HPLC separation using four different solvent gradients and detec
-
ti
on by IT-MS in consecutive reaction monitoring (CRM) mode.
30
Anumberofsta-
tionary phases were evaluated for the extraction of the target compounds (Isolute
ENV+, Oasis HLB, Oasis MCX, Isolute C
8
,IsoluteC
18
,VarianBondElutC
18
,and

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.

MethoddetectionlimitswereinagreementwiththosereportedbyHiltonetal.
30
usingalsoIT-MSinCRMmode.
Stolker et al.
33
reported a combined methodology using LC-(+/-)ESI-MS/MS
and quadrupole-time of ight mass spectrometry (LC-QqToF-MS) for the analysis
of 13 pharmaceuticals, including 4 analgesics (acetylsalicylic acid, diclofenac, ibu-
pr
ofen, and paracetamol), 3 antimicrobials (sulfamethoxazole, erythromycin, and
chloramphenicol), 5 blood-lipid regulators and C-blockers (
fenobrate, bezabrate,
clobric acid, bisoprolol, and metoprolol), and the antiepileptic drug carbamazepine.
ThesampleswereextractedinHLB-MCXSPEcolumn,andtherecoveriesofthe
method were between 60 and 75% for all the compounds except fenobrate, whose
recovery was too low—36%; probably because of its relatively nonpolar character,
the selected extraction conditions were not optimum for this compound.
33
Other
authors
24
reported higher recoveries for fenobrate—more than 90% using LiChro-
lute100RP-18asastationaryphasetoextractthiscompoundfromwaterssamples.
Acetylsalicylicacidpresentedarecoveryof195%.Thiscouldbeexplainedbythe
phenomenon of ion enhancement for this early eluting compound. LC-QqToF-MS
wasusedonlyforconrmatorypurposes.
© 2008 by Taylor & Francis Group, LLC
Advances in the Analysis of Pharmaceuticals in the Aquatic Environment 67
In another study,
34

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

or ethyl acetate-acetone were evaluated. A mixture of ethyl acetate-acetone (50:50)
providedthebestrecoveriesforallcompounds.Regardingtheoptimizationofthe
washing solvent mixture, the authors
35
foundthatupto20%ofmethanolinthewash-
ingsolventdidnotaffectanalyterecoveries,evenforthehighlypolarcompound,
clobricacid.However,signicantanalytelossoccurredforalltargetcompounds
whenthemethanolcontentwasincreasedto50%.Inordertobemorecautious,they
used methanol/water (10:90) as a washing solvent for all subsequent experiments.
Relative recoveries (corrected with internal standard) were between 71 and 118%.
Themethoddetectionlimitswerebetween1and10ng/L(i.e.,twoordersofmagni
-
tu
dehigherrelativetothosereportedbyWeigeletal.
34
).
2.3 DETERMINATION OF DRUGS ACCORDING TO THEIR CLASS
Inthissectionacomprehensivereviewofmethodsdevelopedforspecictherapeutic
classes is provided. A large number of publications dealing with the determination of
drugs in water using advanced mass spectrometric techniques have been published.
TheextractionandpreconcentrationtechniquesinvolveSPEinwhichmanydiffer
-
en
tsorbenttypes,elutingschemes,andsolventswithorwithoutionpairingreagents,
buffers, and modiers were used. Discussion here will be limited to aqueous sam
-
pl
esbecauseChapter3willbedevotedtoissuesrelatedtoextractionandanalysisof
solid-bound pharmaceuticals.
2.3.1 ANALGESICS AND ANTIINFLAMMATORY DRUGS

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

,river,anddrinkingwater.ThemethodconsistedofSPEusingRP-C
18
followed
by methylation of the carboxylic groups with diazomethane, acetylation of phenolic
hydroxyl groups with acetanydride/triethylamine, and determination by GC-MS and
GC-IT-MS/MS. The MQL down to 10 ng/L were achieved in wastewater efuents
aswellinriverwaterbyGC-MSanddownto1ng/LusingGC-IT-MS/MS.Other
authorsalsousedGC-IT-MS/MSforthedeterminationofeightacidicpharmaceu
-
ti
cals in water by SPE on RP-C
18
.In-portmethylationintheGCusingtrimethyl-
sulfoniumhydroxide improved the detection limits such that concentrations in the
ng/L range could be achieved.
38
Recently LC-MS/MS have become the common
methodology for the separation and detection of the analgesic, antiinammatory,
andbloodlipidregulatordrugs.Acidicdrugs,mostofwhicharederivativesofphe-
ny
laceticacid,oftenhavebeendetectedundernegativeionizationmodeconditions,
anddeprotonatedmolecules[M-H]
-
were chosen as a precursor ions.
24
Typically
theyshowed,tovaryingdegrees,thecharacteristictendencytoloseCO
2
,leading
to a benzyl anion that is stabilized by conjugation with the aromatic ring and a lim-

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

growth.
40
Antimicrobialsareofaparticularconcernbecausetheirwideapplication
has led to the selection of resistant bacteria that can threaten the effectiveness of
antimicrobials for the treatment of human infections. Antibiotic-resistant bacteria
reach the environment through animal and human waste, which could transfer the
resistancegenestootherbacterialspecies.
41
Some studies have reported the occurrence of antimicrobials in sewage sludge of
WWTPs
42
and surface waters
43
commonly using LC-MS/MS. For example, sulfon-
amides and tetracyclines, the two most frequently analyzed families of antimicrobi-
a
l
s,aredetectedcommonlybyLC-(+)ESI-MS/MS.Fortetracyclines,theproduct
ions produced from the protonated molecule [M+H]
+
at low collision energies were
the losses of H
2
OandNH
3
(from tetracyclines containing a tertiary OH moiety at
C-6) to nally give abundant [M-H
2
O-NH
3

+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

at elevated probe temperatures oxalic acid decomposes to carbon dioxide and water.
In the absence of stable isotope-labeled surrogate standards for quantitation, they
prepared a series of standard solutions by spiking the analytes into ltered efuent
samples and extraction by SPE and analysis by LC-ESI-MS/MS. Analytical data
from the spiked samples were used to construct standard calibration curves for quan
-
tifying the analytes in the unspiked samples. These calibration curves compensated
forbothvariationsintheSPErecoveriesandmatrixeffectsthatcaneithersuppress
or enhance signals with LC-ESI-MS/MS instrumentation.
Another multianalyte method for the determination of 18 antimicrobials in water
using SPE (mixed LiChrolute EN and LiChrolute C
18
materials) or freeze drying (100
mL of water) and LC-(-)ESI-MS/MS for all the compounds except for chloramphen-
i
c
ol in positive mode was described.
46
Theanalytesbelongedtodifferentgroups
ofantimicrobialssuchaspenicillins,tetracyclines,sulfonamides,andmacrolide
antibiotics. Except for dehydrated-erythromycin, trimethoprim, and tetracycline,
recoveries from freeze drying were greater than 80%, and for SPE method recover
-
i
e
s were slightly lower than for the previous methodology. Tetracyclines were not
recovered in the SPE methodology because Na
2
EDTAwasnotaddedtothewater.
© 2008 by Taylor & Francis Group, LLC

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

basicandacidicpH;however,tetracyclinesinacidicpHwerebetterrecovered.The
authorsappliedaniontrapdata-dependentscanningmethod,whichsimultaneously
collected full scan and MS/MS data for unequivocal identication of target analytes.
Other authors
47
also reported the determination of 13 antimicrobials, sulfonamides,
andtetracyclines,insurfacewatersusingSPE(OasisHLB)andLC-(+)ESI-IT-MS
butinCRMmode.Fortheenrichmentofthewatersamples,pHbetween2and3was
usedbecausetetracyclinesarenotstableatpH<2.Theycheckedtheeffectofthe
columndiameter,owrate,andtemperature(15°C,25°C,35°C)onthepeakshape
in the chromatographic separation, and 15°C was the temperature of choice because
thislowertemperatureresultedinabetterpeakshapesymmetry.Todeterminethe
matrix effects, the authors checked the performance of the internal standard sima
-
to
neintheESIinterfaceduring7months.Nosignicantvariabilityinthepeakarea
during this time was observed, concluding that this standard was not affect by the
matrix suppression. The authors checked the ion suppression of tetracyclines and
sulfonamides, and the surface-water matrix effects were signicant when measuring
tetracyclines but not sulfonamides. Although the limits of detection and quantitation
dependonthevolumeofsampleextracted,thecomplexityofwatermatrices,and
injectionvolumeofextract,similarlimitsofquantitationwereobtainedinthese
studies.
43,47
To correct matrix suppression and losses in the SPE method, a method
for13antimicrobialsandthemetaboliteN4-acetyl-sulfamethoxazoleinwastewa-
te
r using ve labeled internal standards was reported.
42
The method combined SPE

(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

For the analysis of some statins, a class of compounds belonging
to the blood lipid regulators like the brates, negative ionization mode is usually
the method of choice. Miao and Metcalfe
49
reported the analysis of some statins
(atorvastatin, lovastatin, pravastatin, and simvastatin) in waters with SPE and LC-
ESI-MS/MSinbothnegativeandpositivemode.LC-(+)ESI-MS/MSwithmethyl-
am
monium acetate as an additive in the mobile was more sensitive than negative
mode for all compounds. Protonated atorvastatin and methylammonium-adducted
lovastatin,pravastatin,andsimvastatinwereselectedasprecursorions,andproduct
ions were detected by MRM. The instrumental detection limits of atorvastatin, lov
-
as
tatin, pravastatin, and simvastatin were 0.7, 0.7, 8.2, and 0.9 pg, respectively, and
themethoddetectionlimitswerebetween0.1and15.4ng/L.
Carbamazepine belongs to the group of antiepileptic drugs where it represents
thebasictherapeuticagentfortreatmentofepilepsy.Thiscompoundisoneofthe
most frequently detected pharmaceuticals in wastewater and river water. This drug is
generally analyzed with ESI interface in positive mode due to the higher sensitivity
foundcomparedwithAPCI.Theprotonatedmoleculeundergoesfragmentationto
thelossof43Da,whichcorrespondstothecarbamoylgroup.
Carbamazepine undergoes extensive hepatic metabolism by cytochrome P450
system. Thirty-three metabolites of carbamazepine have been identied from
human and rat urine.
50
Aquantitativemethodforsimultaneousdeterminationof
carbamazepine and 5 of its 33 metabolites (10,11-dihydro-10,11-epoxycarbamaze-
pine, 10,11-dihydro-10,11-dihydroxycarbamazepine, 2-hydroxycarbamazepine, 3-
hydroxycarbamazepine, 10,11-dihydro-10-hydroxycarbamazepine) was reported.

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

andifosfamideweredetectedinhospitalefuents
52
as well as WWTP inuents and
efuents.
53
Apparently, ifosfamide reaches surface waters because no biodegrada-
tion occurs in WWTPs. One study determined the expected concentration of ifos-
fa
mideinGermansurfacewatersat8ng/Lbasedondatamodeling.
53
Buerge et al.
54
developed a highly sensitive analytical method based on SPE (macroporous poly-
st
yrene-divinylbenzene) and LC-(+)ESI-MS/MS for the determination of the anti-
tu
moraldrugsifosfamideandcyclophosphamideinwastewaterandsurfacewaters.
Recoveriesrangedfrom74to94%forcyclophosphamideandfrom75to102%for
ifosfamide.Methoddetectionlimitsrangedfrom0.002to0.1ng/Lingroundwater
andbetween0.2and2ng/Lforwastewaters(
Table 2.1).Mahniketal.
55
reported a
method to determine anthracyclines in hospital efuents by SPE (C
8
) combined with
LC-uorescence detection. To extract the anthracyclines from water, bovine serum
wasaddedinordertohavelinearstandardcurvesbecausethesecompoundsadsorb
on surfaces. The authors obtained quantitative recoveries >80% and limits of quan
-

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

The authors recom-
mended the use of LC-(+)ESI-MS/MS for the analysis of these polar molecules in the
environment, because the derivatization of the hydroxyl groups required for GC-MS
analysis was incomplete.
2.3.6 ESTROGENS
Recently,amultitudeofchemicalshaveshowntoactasendocrinedisruptersdisturb-
ing the hormonal systems of aquatic organisms. These compounds can be classied
into naturally occurring and xenobiotic compounds.
56
Naturalsubstancesinclude
sex hormones (estrogens, progesterone, and testosterone) and phytoestrogens, while
© 2008 by Taylor & Francis Group, LLC
Advances in the Analysis of Pharmaceuticals in the Aquatic Environment 73
xenobiotic endocrine disruptors include synthetic hormones, such as the contracep-
tive 17B-ethinylestradiol and man-made chemicals and their by-products (e.g., pes-
ticides and ame retardants). Natural hormones and contraceptives are endocrine
disruptors with effective concentrations at low ng/L levels.
57
Estrogens encompass a
groupofcompoundswithsteroidstructurescontainingphenolicandsometimesali-
phatic hydroxy groups. Both natural and synthetic can be analyzed simultaneously
because of their physicochemical properties.
A sensitive method using SPE (combination of RP-C18 and LiChrolute EN)
andGC-IT-MS/MSforthequanticationofsevenestrogensinsewagesamplesand
riverwaterwasdeveloped.
57
Recoveries of the analytes in groundwater after SPE,
cleanup, and derivatization generally exceed 75%. Method limit of quantication in
differentwaterswerebetween0.5ad1ng/L(Table 2.1). The c
onrmation provided

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.

62
optimizedaprotocolforthe
determinationofsixICM(diatrizoate,iomeprol,iopamidol,iopromide,iothalamic
acid,andioxithalamicacid)inaqueousmatriceswithSPEandLC-(+)ESI-MS/MS.
SeveralSPEsorbents(LiChroluteRP-C
18
,LiChroluteEN,combinationofthecar-
tridgesRP-C
18
andEN,andIsoluteENV+)weretested.SPEusingIsoluteENV+mate-
rialprovedtobethemethodofchoicesincehigherrecoveriesandlowerquantitation
limitswereachieved(Table2 .1). Regarding t
he matrix suppression effects in the ESI
interface, the authors studied the recoveries of the ICM in different water samples.
Thankstotheadditionofasurrogatestandard,therelativerecoveriesfromsurface-
© 2008 by Taylor & Francis Group, LLC
74 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems
water samples were generally quantitative. However, analyzing the WWTP efuents,
some matrix inuences for ioxithalamic acid were present, reducing its recovery to
5%.Adoptingthisapproach,severalauthorsreportedsimilarmethodperformance.
12
Putschew et al.
63,64
proposedanotheranalyticalmethodbasedonasequen-
tial SPE for the isolation of ve ICM (diatrizoate, iopromide, iotrolan, iotrolan,
iotroxin acid) and their possible metabolites (ipha and phipha) using LiChrolute EN
and Enviro-Carb as extraction materials and LC-(+)ESI/MS-MS as the analyzing
method. The recoveries for all compounds were higher than 70°%; only the ionic
compounds were recovered at levels of between 55 and 61%. The method detec
-

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

structures the protonated precursor ion usually produces the loss of H
2
OandHI.
66
Theapplicationof(–)-ESImodewasparticularlyattractiveforthoseICMbearing
afreecarboxylicacid,thoughnonionicICMhavealsobeenreportedtoproduce
[M–H]
–
ions.
67
Negative ionization was also successfully applied to the compound-
classspecicdetectionofICMallofwhichcarryingaromaticiodine.Operatingthe
ionsourceatahighconevoltageledtoin-sourcefragmentationofthedeprotonated
parent molecules resulting in the formation of the diagnostic iodide anion that was
monitoredatm/z127duringaselectedionmonitoringacquisition.
67
Although this
approachsufferedselectivityascomparedwiththeMRMmodetraditionallyusedin
triple quadrupole instruments, monitoring a single ion during the entire chromato
-
gr
aphicrunaddedsomesensitivitytothetechnique.
2.3.8 DRUGS OF ABUSE
Althoughmanysubstancesareincludedinthisgroup(heroin,tetrahydrocannabinol,
cocaine, phencyclidine, LSD, psilocybin, and mescaline), only one study about the
occurrenceofcocaineintheaquaticenvironmenthasbeenpublished.Zuccatoet
al.
16
developedamethodtoanalyzecocaineanditsmainhumanmetabolite,ben-
zoylecgonine, in surface and wastewaters using an SPE (Oasis HLB) method and

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

and phenobarbital and the metabolite of the antiepileptic
primidoneintheefuentofaWWTP.
71
IntheWWTPthesetwodrugswerefoundat
concentrations of 30 and 1000 µg/L, respectively.
Recently, Peschka et al.
72
reported the study of the occurrence of some barbi-
turates, including butalbital, secobarbital, pentobarbital, hexobarbital, aprobarbital,
andphenobarbital,insurfaceandgroundwaters.AmethodusinganSPE(Oasis
HLB)andGCwasdeveloped,showingmethodlimitsofdetectiondownto1ng/L.
Good recoveries of selected barbiturates were obtained from spiked surface water
samples, with values between 64 and 105%, and groundwater and wastewater efu
-
en
tswith,ingeneral,slightlylowervaluesrangingfrom67to104%and52to105%,
respectively (
Table 2.1). The d
rugs were found in surface and groundwaters, indicat-
ing a strong recalcitrance of these compounds, which had been used at high levels
in 1960.
2.4 CONCLUSION
Drugs and their metabolites are present in the environment at low levels, and the use
of advanced analytical methods that afford low limits of detection and high selec-
ti
vityhasallowedforthedetectionofthematlowng/Llevels.Moreover,thanksto
these methodologies, the number of analytes detected has increased considerably
duringthelastdecade.Theproperchoiceofanextractionandcleanupmethodology
forthedeterminationoftheseemergingcontaminantsisanimportantstepinthe
development of an analytical method, because the success of the analytical determi

-
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

-
zat
ion of the parameters of the extraction and cleanup of the sample, including sta-
tionaryphases,andwashingandelutingsolvent,canbeafeasibleapproachforthat
purpose.Dilutionofextractshasalsobeenreportedasaneconomicmethodology
forreducingmatrixeffects.Finally,theuseofisotopedilutiontocompensatematrix
effects also has been discussed in this chapter.
Multiresidue methods offer advantages in terms of providing a more comprehen-
si
ve picture of the occurrence and fate of the contaminants in the environment exam-
in
ed. In addition, the simultaneous determination of a large number of analytes by a
single method represents a less time-consuming and hence more economic approach
as compared with applying several drug-class specic protocols.
ACKNOWLEDGMENTS
The work described in this article was supported by the EU Project (EMCO-INCO-
CT-2004-509188) and by the Spanish Ministerio de Educación y Ciencia Project
EVITA (CTM2004-06255-CO3-01). This work reects only the author’s views, and
theEuropeanCommunityisnotliableforanyusethatmaybemadeoftheinforma-
ti
oncontainedtherein.SPacknowledgesapostdoctoralcontractfromI3PProgram
(ItinerarioIntegradodeInserciónProfesional),conancedbyCSICandEuropean
Social Funds.
REFER ENCES
1. Daughton, C. and Ternes, T.A. 1999. Pharmaceuticals and personal care products in the
environment: agents of subtle change? Environ. Health Perspect. 1
07:907.
2. Daughton, C
. 2004. Non-regulated water contaminants: emerging research. Environ.
Impact Assess. Rev. 2

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.
2002.
Hum. Ecol. Risk. Assess. 8
:657.
6. E
rickson, B.E. Analyzing the ignored environmental contaminants. 2002. Environ.
Sci. Technol. 3
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.
9. Petrovic, M., Hernando, M.D., Diaz-Cruz, M.S., and Barceló, D. 2005. Liquid chro-

matography-tandem mass spectrometry for the analysis of pharmaceuticals residues in
environmental samples: a review.
J. Chromatogr. A 1
067:1.
10. K
olpin, D.W., Furlong, E.T., Meyer, M.T., Thurman, E.M., Zaugg E.D., Barber, L.B.,
and Buxton, H.T. 2002. Pharmaceuticals, hormones and other organic wastewater con-
ta
minantsinU.S.streams,1999–2000:anationalreconnaissance.Environ. Sci. Tech-
nol.
36:1202.
11. P
érez, S., Eichhorn, P., and Aga, D.S. 2005. Evaluating the biodegradability of sul-
famethazine, sulfamethoxazole, sulfathiazole and trimethoprim at different stages of
sewage treatment.
Environ. Toxicol. Chem.
24:1361.
12. C
arballa,M.,Omil,F.,Lema,J.M.,Llompart,M.,García-Jares,C.,Rodríguez,I.,
Gómez, M., and Ternes, T. 2004. Behavior of pharmaceuticals, cosmetics and hor-
mon
esinasewagetreatmentplant. Water Res.,
38:2918.
13. G
lassmeyer, S.T., Furlong, E.T., Kolpin, D.W., Cahill, J. D., Zaugg, S. D., Werner, S. L.,
Meyer, M.T., and Kryak, D.D. 2005. Transport of chemical and microbial compounds
from known wastewater discharges: potential for use as indicators of human fecal con
-
ta
mination. Environ. Sci. Technol. 3

9:5157.
14. Heb
erer, T. 2002. Occurrence, fate, and removal of pharmaceutical residues in the
aquatic environment: a review of recent research data. Toxicol. Lett. 1
31:5.
15. C
alamari, D., Zuccato, E., Castiglioni, S., Bagnati, R., and Fanelli, R. 2003. Strategic
survey of therapeutic drugs in the rivers Po and Lambro in northern Italy. Environ. Sci.
Technol.37:1241.
1
6. Z
uccato, E., Chiabrando, C., Castiglioni, S., Calamari, D., Bagnati, R., Schiarea, S.,
andFanelli,R.2005.Cocaineinsurfacewaters:anewevidence-basedtooltomonitor
communitydrugabuse.
Environ. Health: A Glob. Acc. Sci. Source 4
:14.
17. Q
uintana, J.B., Weiss, S., and Reemtsma, T. 2005. Pathways and metabolites of micro-
bial degradation of selected acidic pharmaceutical and their occurrence in municipal
wastewater treated by a membrane bioreactor.
Water Res.
39:2654.
18. K
alsch, W. 1999. Biodegradation of the iodinated X-ray contrast media diatrizoate and
iopromide. Sci. Total Environ. 2
55:143.
19. P
érez, S. and Barceló, D. 2007. Application of advanced mass spectrometric techniques
in the analysis and identication of human and microbial metabolites of pharmaceuti-
ca

ls in the aquatic environment. Trends Anal. Chem.
26:494.
20. T
ernes, T.A., Bonerz, M., and Schmidt, T. 2001. Determination of neutral pharmaceu-
ticals in wastewater and rivers by liquid-chromatography-electrospray tandem mass
spectrometry.
J. Chromatogr. 9
38:175.
© 2008 by Taylor & Francis Group, LLC