81
3
Sample Preparation
andAnalysisofSolid-
Bound Pharmaceuticals
Christine Klein, Seamus O’Connor,
Jonas Locke, and Diana Aga
3.1 INTRODUCTION
Pharmaceuticals and personal-care products used by humans are often excreted or
washed down drains to wastewater treatment plants, where they can be bound to
particulateinsludgeordischargedintolocalwatersandeventuallybindtosedi-
ments. Similarly, antibiotics and hormones that are used in farm animal operations
canbecomeboundtomanure,soilthatisamendedwiththismanure,andalsoon
air particulate originating from those farms. When pharmaceuticals are bound to
particles, it is less likely that they will undergo biotransformation. However, these
compounds can desorb and become more bioavailable should conditions change,
makingthisprocessfavorable.Therefore,itisimportantforresearcherstoreport
Contents
3.1 Introduction 81
3.2 Matrices of Solid-Bound Pharmaceuticals 82
3.3 Sample Extr act ion Tech n iques 83
3.3.1 Solid–Liquid Extraction 86
3.3.2 Sonication-Assisted Extraction 86
3.3.3 Pressurized Liquid Extraction (PLE) 87
3.3.4 Microwave-Assisted Solvent Extraction 88
3.3.5 Superc r itical Fluid E xt ract ion 89
3.3.6 Matrix Solid Phase Dispersion 89
3.4 Sample Cleanup Techniques 90
3.4.1 Solid Phase Extraction 90
3.4.2 Molecularly Imprinted Polymers 91
3.4.3 Size Exclusion C hromatography 93

extensive these procedures are, the greater the possibility becomes for analyte loss.
Additionally, because the levels of pharmaceuticals are low, these samples will often
need to be preconcentrated in order to be detectable even by the most modern instru-
me
ntation. Unfortunately, this also leads to the concentration of interferences, which
are generally much more abundant in a sample than the analytes themselves.
The extent to which samples containing solid-bound pharmaceuticals are
manipulated, and the best overall analytical method used, depends on several fac-
to
rs.Aswhenpreparingmostenvironmentalsamples,ananalystmustbalancethe
advantages and disadvantages of extraction, cleanup, and concentration techniques.
Compromises are often made, and these decisions are ultimately driven by factors
such as the required detection limit for the purpose of the study, analytical instru-
me
ntation available, the amount and type of extracted material that contaminates a
sample, and the concentrations of analytes present in the samples. This chapter will
review various sample preparation strategies employed in the analysis of pharma-
ce
uticalsinsoil,manure,andsludgeanddiscusstheadvantagesandlimitationsof
each technique.
3.2 MATRICES OF SOLID-BOUND PHARMACEUTICALS
Solid-boundpharmaceuticalshavebeenfoundinmatricesrangingfromhousehold
and farm dust to the sediments that receive treated wastewater. Assessing the chemi-
calcompositionofasampleandknowledgeofpropertiessuchaspolarityandbind-
ingsitescanhelpananalystdeterminethebestextractionmethodtodesorbtheir
analytefromthematrixandbestcleanupmethodtoremoveinterferingmatrixcom-
ponents.Additionalsamplepreparationstepssuchassampledrying,eitherbyairor
© 2008 by Taylor & Francis Group, LLC
Sample Preparation and Analysis of Solid-Bound Pharmaceuticals 83
freeze-drying methods, or mechanical separation by sieving and grinding must be

5
It also has microorganisms and
exocellularmaterialandresiduesoriginatingfromwastewater(forexample,paper
plant residues, oils, fats, and fecal material).
6
Sludgesfromdifferentsourcescan
have enormous compositional variation, which necessitates validation of the extrac-
ti
on efciency for each sludge source.
7
Sediment is comprised of the particulate in surface waters that settles to the
bottom of the water column or remains suspended and transported in waterways.
Sediment particles have both organic and inorganic fractions, which can contain
humic material; metal oxides such as iron and manganese; and also trace metals,
silicates, sulphides, and minerals.
8
Theparticlesizeofsedimentsisoftenindicative
ofitscomponentsandwilldeterminethetypesofcompoundsthataresorbedtoit;
therefore, it often needs to be sieved during its preparation before it is extracted for
contaminants.
So
il is made from eroded earth that is mixed with decayed plant and animal tis-
su
es.Itcontainsmostlyorganiccarbon,inorganicclays,andsand.Pharmaceuticals
arefoundinsoilwhenitisamendedwithsludgesandmanuretogi
ve it more nutrient
content in the form of carbon and nitrogen. Like sediment, soil needs to be sieved
before analysis.
3.3 SAMPLE EXTRACTION TECHNIQUES
Pharmaceuticals that are bound to solids must be removed, or extracted, from these

Butevenwhenpharmaceuticalshavehighwatersolubility,Kdmaybehighdueto
interactions other than hydrophobic.
9
Pressurized liquid extraction (PLE) and super-
criticaluidextraction(SFE)techniquescanleadtohigherextractionefciencies
relative to traditional solid–liquid extraction. Solvent modiers such as acids or bases
are sometimes added to extraction solvents to increase the solubility of analytes in
the extraction solvents and to improve extraction efciencies. Some compounds such
astetracyclinesareknowntoformcomplexeswithdi-andtrivalentcationsinthe
clay minerals or to hydroxyl groups at the surface of soil particles.
10–12
Hence, com-
plexingagentssuchasethylenediaminetetraaceticacid(EDTA)areoftenaddedto
the extraction buffer to improve percent extraction recovery.
13
Thecontacttimeoftheanalytewiththesolidmatrixpriortoextractionisan
important parameter to consider when validating and optimizing extraction proce-
d
u
res. It has been shown for 17B-
estradiol a
nd sulfonamide antibiotics that the longer
thecontacttimebetweenthesoilandtheanalytes,thelowerthepercentextraction
recoveries obtained.
14,15
In addition, when short contact time between the solid and
theanalytewasallowedpriortoPLEextraction,temperaturehadlittleeffectonthe
extraction efciencies of the spiked soils. However, when 17 days of contact time
wasallowed,anincreaseinextractiontemperaturesignicantlyimprovedpercent
recoveries.

e biodegradation when evaluating extraction methods. For instance, the extraction
recoveries for ibuprofen were improved from 25 to 94%, and for trimethoprim from
68 to 86%, when the solid samples were rst autoclaved before fortication with the
analytes,demonstratingtheinuenceoflivemicrobialcommunityontheamount
ofrecoveredpharmaceuticals.Itispossiblethatduringthecontacttimeof14hours
used in the study, the microorganisms have either incorporated the pharmaceuticals
into the organic matter content of the soil or have degraded the pharmaceuticals into
other compounds that were not monitored by the method. The adsorption isotherms
for these compounds in sediment remained unaltered by the autoclaving process,
despite the potential effects of autoclaving on the sediment, organic matter, and cat
-
i
o
n-exchange capacity.
16
Tetracyclines and hormones (such as estrogens) that are introduced into the envi-
ronment present unique challenges that are not encountered in other biological or
food samples. For instance, the strong interaction of tetracyclines with natural organic
matter and with clay components in soil can lead to poor extraction efciencies and
large variability in percent recoveries.
17
While tetracyclines are fairly polar (Kow
0.8), the zwitterionic character of these compounds causes them to complex with ions
TABLE 3.1
Tetracycline Recoveries in Soil Using Accelerated
Solvent Extraction (ASE) and Solid Phase Extraction
(SPE) Cleanup (n = 3)
Spiking Concentration
Tetracycline/Soil
Tetracycline

or more typically the extract volume is reduced to facilitate analysis. This can be
achieved through several different methods and is often dependent on the equipment
available and the analyte being examined. Solid–liquid extraction is simple and cost
effective,sincetheequipmentneededisminimal.However,onemajordrawback
is the relatively large amount of solvent used for this technique compared with the
other techniques discussed below.
Solid–liquidextractionhasbeenusedtoexamineavarietyofpharmaceuticals
in environmental solids. Steroid estrogens, such as estradiol, estrone, estriol, and
ethinylestradiol, have been extracted from sediment and sewage sludge with percent
recoveryrangingfrom61to71%.
18
In another study, the effect of solvent composi-
tionwasexaminedforextractingtetracyclinesfromsediment,anditwasfoundthat
higher citric acid concentration (0.1%) in the solvent improved extraction efcien
-
ci
es up to 105%. However, as the concentration of the chelating agent Na
2
EDTA
increased, the extraction efciency decreased.
19
This conicts with other reports that
suggest that the addition of EDTA into the extraction solvent improves extraction
efciency by releasing metal-complexed tetracyclines.
20
Solid–liquidextractionhasalsobeenusedtodeterminetheconcentrationsofthe
antibioticstylosin,sulfamethazine,chloramphenicol,andtetracyclinesfromthedust
foundinananimalconnementshelter;however,therecoverieswerenotreported.
3
Information on recoveries would have been interesting because the concentration in

and sludge,
7
but primarily in
river sediments.
16,25–28
Inastudythataimedtodetermineantibioticsinpigslurry,sonicationwasused
with a solvent system composed of methanol, McIlvane buffer, and EDTA. The
recoveriesforoxytetracyclineandsulfachloropyridazinerangedfrom77to102%
and58to89%,respectively,usingaconcentrationrangeof1to20mg/L.Asimi
-
la
r extraction method (with methanol added in the solvent) was used to extract soil
thatwasspikedatconcentrationsrangingfrom0.2to0.5ug/g.Therecoverieswere
reportedas27to75%,68to85%,and47to105%foroxytetracycline,sulfachloro
-
pyridazine,andtylosin,respectively,infourdifferenttypesofsoils.Ingeneral,lower
recoveries were observed in soils with higher clay and organic carbon content, espe
-
ci
allyforoxytetracyclineandtylosin,whichhavehigherKdvalues.
24
However, the
spiking levels used in these studies are orders of magnitude above environmentally
relevant concentrations, and therefore these methods may not be applicable to real
environmental samples. Additionally, no mention is made regarding the contact time
used during the recovery studies; this ignores the effect of aging on the extractability
of pharmaceuticals from soil. Similarly, estrogen analysis was attempted in freeze-
driedsolidsfromhoglagoonsamplesusingsonicationandamethanol/acetonemix
-
tu

Another parameter that is often optimized in PLE is the number of
extraction cycles and the need to prewet the soil.
29
However, it appears that for some
compounds, such as uoroquinolones, the best extraction recovery is obtained with
no prewetting of the solid sample.
Thermal degradation studies must be conducted when using elevated tempera
-
tu
res in PLE. Typically, they are conducted by spiking the analytes on quartz sand
andextractingthesandbyPLEatvarioustemperaturesettings.Itisimportantto
assess the effect of high temperature on the stability of the analytes because some
pharmaceutical compounds are thermally labile. Another important consideration is
the amount of sample used for extraction. The amount of sample must be minimized
(5 to 10 g) to avoid unnecessary extraction of large amounts of organic matter and
other matrix components.
15
Acoextractedmatrixnotonlyinterfereswiththeanalyte
detection, but could also clog the extraction vessels, as has been observed during
the extraction of 25 g soil.
1
Furthermore, additives and buffers that are used in other
extraction methods can precipitate and clog the lines of the PLE apparatus.
1
PLE has
been used to extract tetracyclines,
1
macrolides, ionophores, sulfonamides, uoroqui-
nolones,
29,30

organic matter.
33
It was suspected that the manganese oxides present in the sediment
have catalyzed this reaction when exposed to microwaves. The study by Morales-
Munoz
35
showedtheimportanceofsolventpHwithrespecttotheanalyteandpKa/
ionizablefunctionalgroups.Whenananalyteisprotonatedordeprotonated,its
solubility in water (used as the extracting solvent) is increased. The study by Prat
34
illustrates the advantages of using MAE over conventional extraction techniques.
For instance, the use of MAE in a 15-minute extraction of uoroquinolone resulted
in approximately 80% recovery, while 1-hour of mechanical shaking resulted in less
© 2008 by Taylor & Francis Group, LLC
Sample Preparation and Analysis of Solid-Bound Pharmaceuticals 89
than 40% recovery. Further, MAE allowed recoveries of greater than 90% using
threeextractioncyclesforuoroquinolonefromsoil.
35
3.3.5 SUPERCRITICAL FLUID EXTRACTION
SFEexploitsthepropertiesofauidwhenelevatedtemperaturesandpressuresare
appliedabovetheircriticalpoint.Becauseasupercriticaluidexhibitsthermaland
physicalpropertiesofbothaliquidandagas,thesurfacetensionisnonexistent,
causing the diffusivity to increase. This affords supercritical uids the ability to
readily penetrate porous and brous solids, including environmental matrices. An
advantageofusingSFEisthatitisoftenconductedusingcarbondioxide;therefore,
largeamountsoforganicwastesarenotgenerated.
The use of SFE affords the user a higher degree of selectivity, with minimum
amount of coextracted matrix obtained relative to the other extraction techniques. For
example, SFE was reported to produce the cleanest soil extracts compared to other
methods, such as PLE.

39–41
Usinganoctadecylsilylderiva-
tizedsilicasolidphase,withoxalicacidandEDTAasmodiers,recoveryfrom
catshtissuewas81%withalimitofdetection(LOD)of50µg/kg.
40
The method
wasslightlymorevariablewhenusingmilksamples,withrecoveriesrangingfrom
64 to 94%.
41
The extraction of steroids such as estradiol, testosterone, and proges-
terone, were compared in poultry, porcine, and beef meats. Using MSPD, extraction
© 2008 by Taylor & Francis Group, LLC
90 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems
efciencies greater than 90% for all three compounds were observed.
42
MSPD has
alsobeenusedtoextracttriclosanandparabensfromhouseholddust,withgood
recoveries,rangingfrom80±5–114±9%overspikingconcentrationsof50ng/g
to 300 ng/g.
2
MSPDtechniqueisnotreadilyadaptabletolargenumbersofsample,however,
becausethemixingofsampleandsolidphaseisdonemanually.Inaddition,the
detection limits reported are slightly higher than what is needed for tetracyclines in
environmental soil residue analysis and are in the high ppt range for steroids, which
maynotbelowenoughforenvironmentalsamples.
3.4 SAMPLE CLEANUP TECHNIQUES
Natural organic matters, such as humic and fulvic acids, present in environmental
samples are coextracted with the analytes and often complicate analytical detec-
ti
on.Theproblemsassociatedwithsampleanalysisduetocoextractedcompounds

selectivitythroughchoicesinbothstationaryphaseandelutionsolvent,andability
to automate extraction
.Thes
tatio nary phase is available in reversed phase, normal
© 2008 by Taylor & Francis Group, LLC
Sample Preparation and Analysis of Solid-Bound Pharmaceuticals 91
phase, and ion exchange mode, and in cartridge, disk, pipette tip, and microtiter plate
format.
The
SPEprocesscanbebestdescribedashappeninginfoursteps.Therstis
aconditioningstep,wherethesorbentis“wetted.”Solventsareusedtorinsethe
sorbent and to allow contact of the analytes with the solid phase. The next step
involves the “loading” of the sample onto the cartridge. The sample is passed through
the cartridge by pressure differential. In the third step the cartridge is washed to
clean any residual matrix left behind on the sorbent. Finally, a strongly eluting sol
-
v
e
nt is passed through the cartridge and the eluate is collected. SPE can be imple-
me
nted for various classes on compounds and can be used across classes by using
multimodal and mixed phase extractions; “piggybacking” cartridges on one another
andperformingtandemSPE(se
eFigure3.1).F
orexample,astronganionexchange
(SAX),SPEcartridgeintandemwithareversed-phaseSPEresin(suchasthehydro-
phi
lic–lipophilicbalanced,HLB,cartridge)isoftenusedfortheremovalofNOM
fromsoilextracts.Intheanalysisoftetracyclineantibiotics,theuseoftandemSPE
reducestheamountofNOMconsiderably.Figure3.2semiquantitativelyshowsthe

imprinted polymer for the selective removal of tetracyclines from pig kidney tissue
that was selective for several tetracycline analogues.
45
Suedee and colleagues synthe-
sizedanMIPthatwasclassselectivefortetracyclines,whichwassubsequentlyused
in an afnity membrane for the removal of tetracyclines from water.
47
This latter
study demonstrated that the use of MIPs with broad selectivity to isolate tetracycline
degradation products is feasible. However, research is still needed to investigate
whether or not these materials could be used for the selective preconcentration of
tetracyclinesinhighlycomplexmatrices,suchassoilandsludgeextracts.
MIPshavealsobeensynthesizedtoremoveestrogensandestrogeniccompounds
from environmental matrices such as natural waters.
48,49
Estradiol was successfully
© 2008 by Taylor & Francis Group, LLC
92 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems
removedfromdifferentwatersusinganMIPwithrecoveriesof103to104%for
drinking water, pond water, and well water.
48
ThebindingofestrogenstoanMIP
developedtoaidintheanalysisofestrogensinnaturalwaterswaslowerthanthe
binding of estrogens in deionized waters; however, selective binding up to 76 nmol/
mg of MIP was observed in natural waters, and the MIP proved to be reusable for at
least ve uses without any loss of performance.
49
AlongthelinesofanMIP,aselec-
tive technique using antibodies in immunoafnity chromatography has also been
used to isolate estrogens from environmental samples.

Intensity (mAu)
0
200
400
600
800
1000
1200
1400
1600
1800
SAX-HLB (c)
HLB (b)
SAX-tC18 (d)
tC18 (a)
(a)
(b)
(c)
(d)
FIGURE 3.2 UVchromatogramsofastandard1.0%humicacidsaftercleanupwith(a)tC18
SPE,(b)HLBSPE,(c)tandemSAXandHLBSPE,and(d)tandemSAXandtC18SPE.
(a) (b) (c)
FIGURE 3.3 Asolutioncontaininganalytemolecules(a),issurroundedbymonomersthat
bindtotheanalyteandarethenpolymerizedtoformasolid(b).Theanalyteisthenremoved,
andasolidremainswithsitesthatwillbindspecicallytotheanalyte(c).
© 2008 by Taylor & Francis Group, LLC
94 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems
moleculesfreelyeluteinandoutoftheporesandeluteatthetotalpermeationvol-
ume. This makes gel permeation chromatography an excellent technique for remov-
in

matrix components also absorb light at the same wavelength as the analytes and
interfere with the detection. To overcome these challenges, analysts have used LC
with uorescence detection. This technique uses a derivatization step to convert
the analyte to a uorescent conjugate and improve selectivity. However, an 8-hour
reactiontimewasneededformaximumintensityandstabilityofthederivatized
compound.
24
Despite the time required for derivatization, successful detection of
sulfachloropyridazine, oxytetracycline, and tylosine was achieved in the ppb range
in soil and pig slurry samples.
To date, MS detection of pharmaceutical contaminants has become the method
of choice. Analysis of many pharmaceuticals by LC/MS often employs positive
electrospray ionization ((+)-ESI), although negative electrospray ionization, atmo
-
sphe
ricpressurechemicalionization(APCI),andfastatombombardmentionization
have also been used
52–56
utilizing ion-trap and quadrupole mass spectrometers.
57,58
However, ESI is susceptible to ionization suppression or enhancement due to matrix
interferencesbyhumicacids(HA)coextractedfromsoil,sediment,andsludge,or
from mobile phase additives that are used in improving separation.
59,60
In addition,
ionization enhancement has also been shown to occur when using an APCI interface
© 2008 by Taylor & Francis Group, LLC
Sample Preparation and Analysis of Solid-Bound Pharmaceuticals 95
for mass spectrometry.
61

SIM Signal
500 ng Standard
Extract
Extract + 500 ng
Extract + 1500 ng
FIGURE 3.4 Chromatogram of a standard containing 500 ng of EE2 [m/z (–) 295] is com-
paredwithsamplescontainingtheextractoffreeze-driedsludgesolidsspikedwith500ngof
EE2. Initial analysis of the extracted sample is not able to determine if extraction is working
or if matrix effects have cause peak shifting. An additional 500 ng and then 1000 ng of EE2
is added. Ionization suppression due to matrix effects still gives a smaller signal in a sample
containing3xtheEE2asinastandard.(SIM:selectedmonitoring;EE2:Ethynilestradiol.)
© 2008 by Taylor & Francis Group, LLC
96 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems
analytesarefairlypolarandarenotamenabletodirectGC/MSanalysis.Many
pharmaceutical compounds have been derivatized for GC/MS analysis, such as
NSAIDs,
28
triclosan,
2
and estrogens.
7,18,25,63
Like liquid chromatographic methods,
cleanup for these samples also employs SPEandG
PC;howeve r, other methods such
as HPLC fractionation before derivatization are also used.
7,63
While GC/MS analysis
with derivatization can be tedious, it can offer additional selectivity and improve
detectionlimitsrelativetoLC/MSanalysis.
3.6 CONCLUSION

65–72.
5.
Dignac,M F.,Ginestet,P.,Rybacki,D.,Bruchet,A.,Urbain,V.,andScribe,P.,2000.
Fate of wastewater organic pollution during activated sludge treatment: nature of resid-
ua
lorganicmatter.Water Research 37(17): 4185–4194.
6. Degrémont, ed., Water Treatment Handbook, 1
991. Cachan: Lavoisier Publishing.
7. Ternes, T.A., Andersen, H., Gilberg, D., and Bonerz, M., 2002. Determination of estro-
ge
ns in sludge and sediments by liquid extraction and GC/MS/MS. Analytical Chemis-
try 7
4(14): 3498–3504.
8.ElBilali,L.,Rasnussen,P.E.,Hall,G.E.M.,andFortin,D.,2002.Roleofsediment
composition in trace metal distribution in lake sediments. Applied Geochemistry 17
:
1171–1181.
9.
Tolls, J., 2001. Sorption of veterinary pharmaceuticals in soils: a review. Environmen-
tal Science and Technology 3
5(17): 3397–3406.
10. Albert, A. and Rees, C.W., 1956. Avidity of the tetracyclines for the cations of metals.
Nature (
London,UnitedKingdom)177:433–434.
© 2008 by Taylor & Francis Group, LLC
Sample Preparation and Analysis of Solid-Bound Pharmaceuticals 97
11. Sithole, B.B. and Guy, R.D., 1987. Models for tetracycline in aquatic environments. II.
Interaction with humic substances. Water, Air, and Soil Pollution 3
2(3–4): 315–321.
12. Sithole, B.B. and Guy, R.D., 1987. Models for tetracycline in aquatic environments.

and chromatographic/mass spectrometry considerations, incorporating a case study in
method development.
TrAC, Trends in Analytical Chemistry 2
3(10–11): 737–744.
19.Kim,S C.andCarlson,K.,2007.Quanticationofhumanandveterinaryantibiotics
in water and sediment using SPE/LC/MS/MS. Analytical and Bioanalytical Chemistry
38
7(4): 1301–1315.
20. Lindsey, M.E., Meyer, M., and Thurman, E.M., 2001. Analysis of trace levels of sul-
fon
amide and tetracycline antimicrobials in groundwater and surface water using solid-
phase extraction and liquid chromatography/mass spectrometry. Analytical Chemistry
73
(19): 4640–4646.
21. Hyoetylaeinen,T.andRiekkola,M L.,2004.Approachesforon-linecouplingofextrac-
ti
on and chromatography. Analytical and Bioanalytical Chemistry 3
78(8): 1962–1981.
22. SantosJr.,D.,Krug,F.J.,Pereira,M.D.G.,andKorn,M.,2006.Currentsonultrasound-
assisted extraction for sample preparation and spectroscopic analytes determination.
Applied Spectroscopy Reviews 4
1(3): 305–321.
23. Hutchins, S.R., White, M.V., Hudson, F.M., and Fine, D.D., 2007. Analysis of lagoon
samples from different concentrated animal feeding operations for estrogens and estro-
ge
n conjugates. Environmental Science & Technology 41
(3): 738–744.
24. Blackwell, P.A., Holten Lutzhoft, H C., Ma, H P., Halling-Sorensen, B., Boxall,
A.B.A., and Kay, P., 2004. Ultrasonic extraction of veterinary antibiotics from soils and
pig slurry with SPE clean-up and LC-UV and uorescence detection.

tr
ometry. Journal of Chromatography A 1
003: 21–28.
31. Chun, S., Lee, J., Geyer, R., and White, D., 2005. Comparison of three extraction meth-
od
s for 17b-estrdiol in sand, bentonite and organic rich silt loam. Journal of Environ-
mental Science and Health, Part B 4
0: 731–740.
32. Houtman, C.J., van Houten, Y.K., Leonards, P.E.G., Brouwer, A., Lamoree, M.H., and
Legler,J.,2006.BiologicalvalidationofasamplepreparationmethodforER-CALUX
bioanalysis of estrogenic activity in sediment using mixtures of xeno-estrogens. Envi-
ronmental Science & Technology
4
0(7): 2455–2461.
33.Labadie,P.andHill,E.M.,2007.Analysisofestrogensinriversedimentsbyliquid
chromatography-electrospray ionisation mass spectrometry. Journal of Chromatogra-
phy A
1141(2): 174–181.
34. Prat, M.D., Ramil, D., Compano, R., Hernandez-Arteseros, J.A., and Granados, M.,
2006. Determination of umequine and oxolinic acid in sediments and soils by micro-
wa
ve-assisted extraction and liquid chromatography-uorescence. Analytica Chimica
Acta 567(2): 229–235.
3
5. Morales-Munoz, S.L., Luque-Garcia, J.L., and Luque de Castro, M.D., 2004. Continu-
ous microwave-assisted extraction coupled with derivatization and ouimetric moni-
to
ring for the determination of ouroquinolone antibacterial agents from soil samples.
Journal of Chromatography A 105
9: 25–31.

oxytetracycline, tetracycline, and chlortetracycline in milk. Journal—Association of
Ofcial Analytical Chemists 73
(3): 379–384.
42.Gentili,A.,Sergi,M.,Perret,D.,Marchese,S.,Curini,R.,andLisandrin,S.,2006.
High-andlow-resolutionmassspectrometrycoupledtoliquidchromatographyascon-
r
matory methods of anabolic residues in crude meat and infant foods. Rapid Com-
munications in Mass Spectrometry
2
0(12): 1845–1854.
43. Cai, W. and Gupta, R.B., 2004. Molecularly-imprinted polymers selective for tetracy-
cline binding. Separation and Purication Technology 35(3): 215–221.
© 2008 by Taylor & Francis Group, LLC
Sample Preparation and Analysis of Solid-Bound Pharmaceuticals 99
44. Andersson, L.I., 2000. Molecular imprinting: developments and applications in the
analytical chemistry eld. Journal of Chromatography, B: Biomedical Sciences and
Applications
7
45(1): 3–13.
45. Caro, E., Marce, R.M., Cormack, P.A.G., Sherrington, D.C., and Borrull, F., 2005.
Synthesisandapplicationofanoxytetracyclineimprintedpolymerforthesolid-phase
extraction of tetracycline antibiotics.
Analytica Chimica Acta 5
52(1–2): 81–86.
46. Xiong,Y.,Zhou,H.,Zhang,Z.,He,D.,andHe,C.,2006.Molecularlyimprintedon-line
solid-phase extraction combined with ow-injection chemiluminescence for the deter
-
mi
nation of tetracycline. Analyst (
Cambridge, United Kingdom) 131(7): 829–834.

oxytetracycline, tetracycline, and chlortetracycline in water using solid-phase extrac
-
ti
on and liquid chromatography-tandem mass spectrometry. Journal of Chromatogra-
phy A
9
28: 177–186.
52. Blanchower, W.J., McCracken, R.J., Haggan, A.S., and Kennedy, D.G., 1997. Conr-
ma
tory assay for the determination of tetracycline, oxytetracycline, chlortetracylcine
and its isomers in muscle and kidney using liquid chromatography-mass spectrometry.
Journal of Chromatography B 6
92: 351–360.
53. Oka, H., Ikai, Y., Hayakawa, J., Harada, K I., Asukabe, H., and Suzuki, M., 1994.
Improvement of chemical analysis of antibiotics. 22. Identication of residual tetracy
-
cl
ines in honey by frit FAB/LC/MS using a volatile mobile phase. Journal of Agricul-
ture and Food Chemistry
4
2: 2215–2219.
54. Nakazawa,H.,Ino,S.,Kato,K.,Watanabe,T.,Ito,Y.,andOka,H.,1999.Simultaneous
determination of residual tetracyclines in foods by high-performance liquid chroma
-
to
graphy with atmospheric pressure chemical ionization tandem mass spectrometry.
Journal of Chromatography B 7
32: 55–64.
55. Delepee, R., Maume, D., Bizec, B.L., and Pouliquen, H., 2000. Preliminary assays to
elucidate the structure of oxytetracycline’s degradation products in sediments. Deter

-
tion between target drugs and stable-isotope-labeled internal standards in quantitative
liquid chromatography/tandem mass spectrometry.
Rapid Communications in Mass
Spectrometry 1
7: 2815–2821.
61.Dams,R.,Huestis,M.A.,Lambert,W.E.,andMurphy,C.M.,2003.Matrixeffectin
bio-analysis of illicit drugs with LC-MS/MS: inuence of ionization type, sample
preparation, and biouid.
Journal of the American Society of Mass Spectrometry 1
4
:
1290–1294.
62
. Schnitzer, M., 1978. Humic substances: chemistry and reactions.
Developments in Soil
Science 8(Soil Org. Matter): 1–64.
63.Williams,R.J.,Johnson,A.C.,Smith,J.J.L.,andKanda,R.,2003.Steroidestrogens
prolesalongriverstretchesarisingfromsewagetreatmentworksdischarges.
Environ-
mental Science and Technology 37
(9): 1744–1750.
© 2008 by Taylor & Francis Group, LLC