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 3.5 Spe cial Considerations in Sa mple Ana lysis 94 3.5.1 Liquid Chromatography 94 3.5.2 Gas Chromatography 95 3.6 Conclusion 96 References 96 © 2008 by Taylor & Francis Group, LLC 82 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems the fraction of pharmaceuticals that are bound to solids in environmental studies insteadofjustconcludingthatthepharmaceuticalhasbeenbiodegradedorother- wiseremovedfromthesystem. The extent to which these compounds become bound to solids can be character- izedbyacompound’sKd(asolid-waterpartitioningcoefcient)value.SeeChapter 6 for more information on sorption processes. The environmental solids these phar- ma ceuticals can bind to are diverse in composition. Because the degree of binding depends highly on the nature of the sorbent-sorbate interactions, the available meth- ods for extraction of pharmaceuticals from solid matrices vary widely. Hence, the applicabilityofextractionandanalyticalmethodsneedstobeevaluatedfordifferent pharmaceutical compounds, and conditions need to be optimized for various types of solids. Onceanextractionmethodisfoundtobesuitableforremovingsorbedpharma- ce uticalsfromenvironmentalsolids,itisoftennecessarytoperformsomedegreeof cleanuponthesesamplesbeforedetectionandquantication.Environmentalmatri- cessuchasmanure,soil,sludge,andsedimentallcontainnaturalorganicmatter (NOM),whichisgenerallydescribedasapoorlydenedmixtureoforganicsub- stanceswithvariablepropertiesintermsofacidity,molecularweight,andmolecular structure. Many times extraction methods will coextract portions of the environmen- talmatrix,whichcaninterferewithanalysisinavarietyofways.Throughextraction and cleanup, one is able to remove many matrix interferences; however, the more 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 takenintoconsiderationbecauseoftheirlaborintensivenessandeffectonthetime ittakestoprepareasample.Ingeneral,thelessnaturalorganicmattercontentin amatrix,theeasieritistoextractorganicanalytes.Forexample,whencomparing extraction efciencies from different soil types, higher recoveries for tetracyclines (TCs)areo bserved in sandier soil than in soil with more organic matter. 1 The compositions of typical matrices encountered when conducting environmen- tal analysis are described in this section. Air particulate has been found to contain antibacterial agents such as triclosan 2 in household dust and tetracyclines, sulfa- methazine,tylosine,andchloramphenicolinanimalconnementbuildings. 3 The dust from the interior of an animal connement building was analyzed and found to contain approximately 85% organic material, composed of protein (from skin), animal feed, endotoxins, fungi, and bacteria. 4 This composition will vary, depending on the source. Sludges have been found to be contaminated with a variety of compounds ranging from personal-care products and pharmaceuticals, which are washed and usheddowndrains,tohormonesandantibiotics,whichareusedinanimalpro - duction. Wastewater sludge contains many compounds and is primarily organic in nature.Thecompositionofthisorganicportionofsludgeismadeupofsugars,pro - teins, fatty acids, cellulose, and plant macromolecules with phenolic and aliphatic structuresbutisstillnotcompletelycharacterized. 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 efciency 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 solidspriortoanalysis.However,astandardmethodforextractiondoesnotexist, and an extraction procedure must be optimized for the conditions that an analyst encounters. One of the most widely used methods for assessing the efciency of an extractionprocedureisbydeterminingthepercentrecoveryandextractionyieldof amethodthroughspikingexperiments.Inthistypeofexperimentananalystwill © 2008 by Taylor & Francis Group, LLC 84 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems take a sample of the matrix that they want to extract a compound from, add a known amount of that compound, and extract it using the selected method to determine howmuchisremoved.Issuesthatmustbeconsideredwhenperformingthistypeof experimentare(1)solventselection;(2)contacttime;(3)spikinglevel;and(4)pos - s i ble effects on binding/transformation by the microbial community present in the sample.Italsoisimportanttovalidatemethodswhencomparingextractionofdif - fe rent types of solids because efciencies can differ, leading to gross underestima- t i on or overestimation of analyte concentration. Simply adding an internal standard does not excuse the validation of an extraction procedure, because the contact time withthesolidsmaynotbeappropriateforassessingextractionefciency. Solvent selection is probably the most important step in developing an extraction methodforsolid-boundpharmaceuticals.Theanalyteshouldhavehighsolubilityin the extraction solvent in order to desorb the analytes efciently. Many pharmaceuti - calcompounds,suchasantibioticsandhormones,havelowwatersolubilityandare relatively hydrophobic, making it necessary to use organic solvents for extraction. Butevenwhenpharmaceuticalshavehighwatersolubility,Kdmaybehighdueto interactions other than hydrophobic. 9 Pressurized liquid extraction (PLE) and super- criticaluidextraction(SFE)techniquescanleadtohigherextractionefciencies relative to traditional solid–liquid extraction. Solvent modiers 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 efciencies. 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 efciencies of the spiked soils. However, when 17 days of contact time wasallowed,anincreaseinextractiontemperaturesignicantlyimprovedpercent recoveries. 15 Spiking a solid matrix at environmentally relevant concentrations is also impor- tant when determining extraction efciency. It may be tempting for an analyst to spike at higher levels because this can alleviate problems associated with detection. However, this can mislead one into believing that the extraction efciency is higher or more reproducible than it actually is at the lower concentrations typically observed in the natural environment. For instance, in an experiment conducted in our labora - t o ry to nd optimized extraction conditions for tetracyclines from soil, the percent recovery at low concentrations was signicantly higher than at the spiked concen - tr ations.Whensoilwasspiked(n=3)atconcentrationsof100ng/g,therecoveries oftetracyclinesrangedfrom89to92%,withstandarddeviationsatorbelow10% (see Table 3.1). H owever, w hen the spiking levels approached environmentally rel- evant concentrations (below 20 ng/g), exaggerated recoveries (>100%) and very high © 2008 by Taylor & Francis Group, LLC Sample Preparation and Analysis of Solid-Bound Pharmaceuticals 85 standard deviations (>50%) were observed, suggesting signicant matrix interfer- ence that is highly variable. Microbial communities present in the matrix could potentially alter the sorp- ti on of pharmaceutical compounds in soil via biodegradation or biotransformation. Therefore,itisimportanttoensurepropersamplestorageandtoaccountforpossi- bl 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 fortication with the analytes,demonstratingtheinuenceoflivemicrobialcommunityontheamount 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 efciencies 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 Compound Percent Recovery Standard Deviation 100 ng/g TC 91% 3% 100 ng/g OTC 89% 10% 100 ng/g CTC 92% 6% 50 ng/g TC 140% 10% 50 ng/g OTC 151% 5% 50 ng/g CTC 155% 10% 25 ng/g TC 140% 47% 25 ng/g OTC 138% 47% 25 ng/g CTC 171% 50% Note: As spiking level approaches environmentally relevant concentra- tions below 25 ng/g, recovery becomes less reproducible. TC = Tetracycline OTC = Oxytetracycline CTC = Chlortetracycline © 2008 by Taylor & Francis Group, LLC 86 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems present in soil or sludge. Estrogens, on the other hand, are fairly nonpolar (Kow 4.2) andthushavehighsorptiontosolidmatriceswithhighorganicmattercontent. 3.3.1 SOLID–LIQUID EXTRACTION Solid–liquid extraction is the most basic extraction method for solid samples. It involvesmechanicalagitationofamixtureconsistingofthesampletobeextracted andanexcessofsolvent.Thesolventandsampleareextractedforaperiodoftime, typicallylongerthan10minutes.Inatypicalsolid–liquidextraction,thesampleis then centrifuged, and the supernatant is removed and extraction is repeated several times.Thesupernatantsarecombined,atwhichtimethesamplecanbeanalyzed, 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 efcien - ci es up to 105%. However, as the concentration of the chelating agent Na 2 EDTA increased, the extraction efciency decreased. 19 This conicts with other reports that suggest that the addition of EDTA into the extraction solvent improves extraction efciency by releasing metal-complexed tetracyclines. 20 Solid–liquidextractionhasalsobeenusedtodeterminetheconcentrationsofthe antibioticstylosin,sulfamethazine,chloramphenicol,andtetracyclinesfromthedust foundinananimalconnementshelter;however,therecoverieswerenotreported. 3 Information on recoveries would have been interesting because the concentration in dust is typically very low. Although solid–liquid extraction is frequently used in the analysisofawiderangeofpharmaceuticals,itsuseislimitedbecausecompounds thatarestronglysorbedtoasolidmatrixmayneedmorerigorousextractioncondi - t i ons, such as those provided by the other techniques discussed below. 3.3.2 SONICATION-ASSISTED EXTRACTION Insonicationassistedextraction(SAE)samplesaremixedwithanextractionsol- ventandplacedintoasonicationbath.Thesamplemixtureissubjectedtoacoustic vibrations with frequencies above 20 kHz. These ultrasonic waves travel through the sample, leading to expansion and compression cycles in the solvent. The expansion cycles cause a negative pressure in the liquid, and if the amplitude of these waves is strong enough, cavities or bubbles in the solvent can be observed. Upon the col - lapse of these bubbles, localized temperatures and pressures can exceed 5000 K and 1000 atm, respectively, creating shockwaves which in turn increase the desorption © 2008 by Taylor & Francis Group, LLC Sample Preparation and Analysis of Solid-Bound Pharmaceuticals 87 of analytes from the matrix surface. 21 Additionally, the collapse of these bubbles in the presence of suspended particles can lead to asymmetric collapses that form high- speedmicrojetstowardthesolid’ssurface,leadingtoerosionandcleavages.The increase in surface area also improves extraction of analytes. 22 This technology has been used for the extraction of pharmaceuticals and natural hormones from matrices such as manure and soil amended with manure, 23,24 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 re as an extraction solvent. 23 However, extraction recoveries were not reported; hence, no assessment can be made on how successful the sonication method is for estrogen extraction from solids. 3.3.3 PRESSURIZED LIQUID EXTRACTION (PLE) PLE, also known as accelerated solvent extraction (ASE), involves the use of pres- surized extraction vessels at elevated temperatures to achieve efcient extraction of analytes. The sample is placed in an extraction cell with an inert solid dispersant. Thesoliddispersant,suchassandordiatomaceousearth,servesatwofoldpurpose. First,itllstheemptycellvolumetominimizeexcesssolventconsumption,and second, it increases the surface area of the sample that is exposed to the extrac - ti on solvent. The lled extraction cell is then pressurized with extraction solvent andplacedinanoventoincreasethetemperature.Highertemperaturesincrease solubility of analytes in the solvent and decrease viscosity of the solvent, leading to betterpenetrationintointerstitialspacespresentinthesample.Additionally,raising the temperature increases extraction kinetics such as desorption. Elevated pressures also contribute somewhat to increasing solvent contact with the sample, but mainly theyservetokeepthesolventsliquidattheincreasedtemperature.PLEoffersthe advantage in many cases of automation, leading to increased sample throughput. Furthermore, PLE reduces the solvent requirement to extract samples, saving on analysis cost and minimizing organic solvent waste. © 2008 by Taylor & Francis Group, LLC 88 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems In studies that compare the effects of solvent, temperature, pressure, and time using PLE, solvent selection generally has the most signicant effect, followed by temperature. 15,29 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 and estrogens 31,32 fromsoil,sludge,andsediments. 3.3.4 MICROWAVE-ASSISTED SOLVENT EXTRACTION Another emerging technique in environmental analysis is microwave-assisted extrac- tion(MAE).Thistechniqueinvolvestheuseofmicrowavestoheatasampleina closed vessel so that temperatures above the normal boiling point of the extraction solvent can be utilized. The increased temperature improves analyte solubility, extrac - ti on kinetics, and solvent contact (wetting) with the matrix, similar to PLE. The main advantages of MAE include: decreased extraction time, increased sample through - pu tbymeansofautomatedandsimultaneousextractionofseveralsamples,and decreasedsolventconsumptionrelativetosoxhletextraction.Thedisadvantagesto usingMAEforsoilextractioninclude:thermaldecompositionofanalytes,nonselec - ti ve extraction of matrix components, and limited solvent choices. Only microwave- active solvents such as water and methanol can be used; nonpolar organic solvents such as hexane, cyclohexane, and methylene chloride are not useable in MAE. MAEhasbeensuccessfullyusedfortheextractionofestrogensfromsediments 33 and quinolone antibiotics from soils and sediments. 34,35 It should be noted, however, that during extraction, estradiol was oxidized to estrone in sediment c ontaining low 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 threeextractioncyclesforuoroquinolonefromsoil. 35 3.3.5 SUPERCRITICAL FLUID EXTRACTION SFEexploitsthepropertiesofauidwhenelevatedtemperaturesandpressuresare appliedabovetheircriticalpoint.Becauseasupercriticaluidexhibitsthermaland 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. 36 SFE has been used to extract nonsteroidal antiinammatory drugs (NSAIDs) from river sediment, 28 as well as 4-nonylphenol and bisphenol A from sludge. 37,38 TherecoveriesofNSAIDssuchasnaproxenandketopronfrom riversedimentusingSFEwerecomparabletotherecoveriesobtainedusingPLE(78 to 79%) and MAE (81 to 82%). On the other hand, the recoveries of 4-nonylphenol and bisphenol A were disappointing and were lower than the recoveries observed using PLE. However, the only parameter that was altered in SFE was the solvent composition;itmightbepossibletoobtainhigherrecoveriesifotherSFEextraction parameters are optimized. 3.3.6 MATRIX SOLID PHASE DISPERSION Matrix solid phase dispersion (MSPD) is another technique that can be used for extraction of analytes from solid samples. Briey, this technique uses a solid phase sorbent, usually octadecylsilyl silica packing material similar to those used in reversed phase high-performance liquid chromatography (HPLC) and solid phase extraction (SPE). This s olid phase is conditioned with the appropriate solvents and thenmixedwiththesolidsampleusingamortarandpestle.Themixtureisthen transferred to a column, and the analytes are eluted with organic solvents. This extraction procedure offers the advantage of providing direct contact between the solidextractingmaterialsandtheanalytesinthesolidsample.Thelargesurface area of the derivatized silica particles used in MSPD facilitates efcient transfer of analytes from soil to the extracting solid phase. MSPD has been applied in the analysis of tetracycline antibiotics in several food- relatedmatrices,suchasmilkandcatshtissue,buttherehasbeennoreportonits application in the analysis of tetracyclines in soil. 39–41 Usinganoctadecylsilylderiva- tizedsilicasolidphase,withoxalicacidandEDTAasmodiers,recoveryfrom catshtissuewas81%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 efciencies 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 are collectively termed “matrix effects.” Hence, the amount of coextracted natural organicmattermustbeminimizedtoachieveasuccessfulanalysis.Themostwidely used technique to concentrate environmental samples and to reduce matrix effects is SPE. Gel permeation chromatography has also been used to separate proteinaceous andhighmolecularweightmaterialsfromsmallermolecularweighttargetanalytes. Recently,molecularlyimprintedpolymers(MIPs)havebeenusedasselectivesor - bents for SPE. The extent of sample cleanup needed will depend on the susceptibil- it y of the analytical instrument to matrix effects. Therefore, the implementation of a cleanup method must be carefully considered for each analyte, sample type, and instrumentation. 3.4.1 SOLID PHASE EXTRACTION SPEisusedtoseparatecompoundsinasamplebasedontheirpolaritiesandsolu- bilitiesinspecicsolventsandisbasedonprinciplessimilartothoseofliquidchro- mat ography.SPEistypicallyconductedbypassingalarge-volumeaqueoussample throughacartridgepackedwithanappropriatesorbent.Ideally,theanalyteswill sorb to the packing material, while most interfering compounds are unretained and pass through the cartridge. The sorbed analytes are then eluted with a relatively small volume of solvent and collected. The eluate may need to be evaporated to a smaller volume and may be solvent exchanged to an appropriate solvent for further analysis. Ontheotherhand,theSPEproceduremaybedesignedsothattheunwantedmatrix can be captured in the SPE sorbent while the analytes pass through the cartridge and are collected for further concentration. Either way, the main purpose of SPE is the removal of matrix components such as salts and some organic matter, while con - ce ntrating analytes. SPE has replaced many conventional liquid–liquid extraction techniquesduetoadvantagesgainedbyminimizingsolventconsumption,increased selectivitythroughchoicesinbothstationaryphaseandelutionsolvent,andability to automate extraction .Thes tatio nary phase is available in reversed phase, normal © 2008 by Taylor & Francis Group, LLC [...]... through tandem SAX and HLB SPE cartridges The humics are retained by the SAX cartridge and the tetracyclines pass through to the HLB cartridge, where they are retained The SAX cartridge is then removed, and the tetracyclines are eluted from the HLB cartridge, minimizing the concentration of humic acids in the sample removed from different waters using an MIP with recoveries of 1 03 to 104% for drinking water, ... water, pond water, and well water. 48 The binding of estrogens to an MIP developed to aid in the analysis of estrogens in natural waters was lower than the 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 five uses without any loss of performance.49 Along the lines of an MIP, a... 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 32 (3 4): 31 5 32 1 12 Sithole, B.B and Guy, R.D., 1987 Models for tetracycline in aquatic environments I Interaction with bentonite clay systems Water, Air, and Soil Pollution 32 (3 4): 30 3 31 4 13 O’Connor,... Antonic, J and Heath, E., 2007 Determination of NSAIDs in river sediment samples Analytical and Bioanalytical Chemistry (38 7): 133 7– 134 2 © 2008 by Taylor & Francis Group, LLC 98 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems 29 Golet, E.M., Strehler, A., Alder, A.C., and Giger, W., 2002 Determination of fluoroquinolone antibacterial agents in sewage sludge and sludge-treated... 94 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems molecules freely elute in and out of the pores and elute at the total permeation volume This makes gel permeation chromatography an excellent technique for removing proteins and lipids from samples It has been used as a cleanup method for steroid estrogens in sediment and sewage sludge.18 ,32 Humic and fulvic acids are often... M., and Kocourek, V., 2007 Novel approaches to the analysis of steroid estrogens in river sediments Analytical and Bioanalytical Chemistry 38 7(4): 135 1– 136 3 27 Lopez de Alda, M.J and Barcelo, D., 2001 Use of solid-phase extraction in various of its modalities for sample preparation in the determination of estrogens and progestogens in sediment and water Journal of Chromatography A 938 (1–2): 145–1 53 28... with the analytes. 63 In the analysis of ethinylestradiol from a freeze-dried sludge extract, the ionization suppression is remarkable A 5-g sample of freeze-dried sludge was spiked with 500 ng of ethinylestradiol and extracted using methanol and acetone with ASE The extract was subjected to SPE cleanup using C-18 cartridges and analyzed using LC/ MS The sample was compared with a standard containing... tandem SPE reduces the amount of NOM considerably Figure 3. 2 semiquantitatively shows the amount of NOM removal when using both C-18 and HLB cartridges in tandem with SAX The ultraviolet (UV) trace for each type of SPE setup was performed on a soil extract, and the corresponding SPE setup is shown in Figure 3. 2 This indicates that the use of tandem SPE for soil samples removes most of the coextracted... of Chromatography A 1021(1–2): 133 –144 17 Kulshrestha, P., Giese, R.F., Jr., and Aga, D.S., 2004 Investigating the molecular interactions of oxytetracycline in clay and organic matter: insights on factors affecting its mobility in soil Environmental Science and Technology 38 : 4097–4105 18 Gomes, R.L., Avcioglu, E., Scrimshaw, M.D., and Lester, J.N., 2004 Steroid-estrogen determination in sediment and. .. critique of sample preparation and chromatographic/mass spectrometry considerations, incorporating a case study in method development TrAC, Trends in Analytical Chemistry 23( 10–11): 737 –744 19 Kim, S.-C and Carlson, K., 2007 Quantification of human and veterinary antibiotics in water and sediment using SPE/LC/MS/MS Analytical and Bioanalytical Chemistry 38 7(4): 130 1– 131 5 20 Lindsey, M.E., Meyer, M., and . Fate of Pharmaceuticals in the Environment and in Water Treatment Systems removedfromdifferentwatersusinganMIPwithrecoveriesof103to104%for drinking water, pond water, and well water. 48 ThebindingofestrogenstoanMIP developedtoaidintheanalysisofestrogensinnaturalwaterswaslowerthanthe binding. Asolutioncontaininganalytemolecules(a),issurroundedbymonomersthat bindtotheanalyteandarethenpolymerizedtoformasolid(b).Theanalyteisthenremoved, andasolidremainswithsitesthatwillbindspecicallytotheanalyte(c). © 2008 by Taylor & Francis Group, LLC 94 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems moleculesfreelyeluteinandoutoftheporesandeluteatthetotalpermeationvol- ume 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