Quantitative Determination of Antidepressants and Their SelectDegradates by Liquid Chromatography/Electrospray IonizationTandem Mass Spectrometry in Biosolids Destined for LandApplicationLydia M. Niemi, Katherine A. Stencel, Madigan J. Murphy, and Melissa M. Schultz*

Department of Chemistry, 943 College Mall, The College of Wooster, Wooster, Ohio 44691, United States

*S Supporting Information

ABSTRACT: Antidepressants are one of the most widelydispensed classes of pharmaceuticals in the United States. Aswastewater treatment plants are a primary source ofpharmaceuticals in the environment, the use of biosolids asfertilizer is a potential route for antidepressants to enter theterrestrial environment. A microsolvent extraction method,utilizing green chemistry, was developed for extraction of thetarget antidepressants and degradation products from bio-solids, or more specifically lagoon biosolids. Liquid chromatography/tandem mass spectrometry was used for quantitativedetermination of antidepressants in the lagoon biosolid extracts. Recoveries from matrix spiking experiments for the individualantidepressants had an average of 96%. The limits of detection for antidepressant pharmaceuticals and degradates ranged from0.36 to 8.0 ng/kg wet weight. The method was applied to biosolids destined for land application. A suite of antidepressants wasconsistently detected in the lagoon biosolid samples, and thus antidepressants are being introduced to terrestrial environmentsthrough the land application of these biosolids. Sertraline and norsertraline were the most abundant antidepressant anddegradation product detected in the biosolid samples. Detected, individual antidepressant concentrations ranged from 8.5 ng/kg(norfluoxetine) to 420 ng/kg wet weight (norsertraline).

Human pharmaceuticals enter wastewater treatment and,subsequently, the environment, primarily by way of

domestic waste from human excretion or by direct disposal ofunused or expired drugs down the drain.1−3 Pharmaceuticallyactive compounds were not commonly viewed as environ-mental contaminants until the late 1990s, when their activeingredients were found to negatively impact ecosystems at lownanogram per liter concentrations.4 During this time, analyticalmethods such as liquid chromatography/tandem massspectrometry were being developed and optimized to measurepharmaceutically active compounds from the environment,allowing the determination of these chemicals at tracequantities. With over 3200 registered pharmaceutical ingre-dients, including antidepressants,5 on the market today, theoccurrence of pharmaceutically active compounds in theenvironment has become a growing concern to the public aswell as scientific and regulatory communities. Daughton andTernes3 expressed concerns that rising pharmaceutical levels inthe environment could cause irreversible change to ourecological systems. Generally, these pharmaceuticals do notdisplay high acute toxicity because lethal effects typically occurat concentrations greater than 1 mg/L.6 However, organismsmay receive continuous exposure in “pseudopersistent”scenarios where chemical half-lives are exceeded by wastewatereffluent introduction rates,7 therefore justifying the need tocharacterize chronic, sublethal effects of these drugs on

nontarget organisms.8−10 Terrestrial organisms may also beexposed to wastewater contaminants through habitats thatreceive land-applied biosolids and, thus, are susceptible tononlethal behavioral and physiological consequences, warrant-ing further investigation.11−14

Antidepressant pharmaceuticals are one of the most heavilyprescribed pharmaceutical products in the United States;15

currently more than 10% of the U.S. population usesantidepressants.16 Since antidepressants are widely prescribedin the U.S. and are incompletely removed during municipalwastewater treatment,16−21 it is not surprising that thesechemicals are being detected in our waterways.17,19,21 Twostudies have investigated the sorption capacities and basicity ofantidepressants and discovered that antidepressants have a highlikelihood to remain unchanged during wastewater treat-ment.22,23 Kwon and Armbrust22 investigated the sorptioncapacities of five selective serotonin reuptake inhibitors (SSRIs)to sediment and soil and discovered that all the studied SSRIshave high sorption capacities, except for fluvoxamine.Lajeunesse et al.23 found that most antidepressants willpartition into the solid-phase sludge during wastewatertreatment due to their sorption coefficients (log Kd > 4).

Received: April 19, 2013Accepted: July 11, 2013Published: July 11, 2013

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Also, both studies indicated that the basicity of antidepressants(pKa 9−10) increases the sorption of molecules to hydrophobicmatrices.22,23

Since antidepressants have high sorption capacities,22,23 theyare likely to be present in sludge and biosolids as compared toaqueous media in wastewater treatment plants. Thus, land-disposed biosolids, used for soil amendment or fertilizer, couldbe another source for antidepressants into (terrestrial)environments. In the United States, approximately 50% of the6.5 million dry metric tons of sewage sludge were applied tosoils in 2004.24 Of that, 75% of the total mass of land-appliedbiosolids was used on agricultural lands.24 Reports of thepresence of antidepressants in biosolids to date is limited.Kinney et al.25 analyzed biosolids for pharmaceuticals andpersonal care products, including fluoxetine, the only targetedantidepressant in the study, from nine different wastewatertreatment plants, and the average concentration of fluoxetinewas 340 ng/g (dry weight). The U.S. Environmental ProtectionAgency (EPA) conducted an assessment of 145 environmentalcontaminants, including one antidepressant (fluoxetine), inbiosolids collected nationwide between 2006 and 2007.Fluoxetine was found in 94% of the samples.26 Fluoxetine,sertraline, paroxetine, and norfluoxetine were present at mean

concentrations of 171, 458, 62, and 42 ng/g (dry weight),respectively, in archived biosolids (2001) that were analyzed asfive megacomposites representing 94 wastewater treatmentplants in 32 states.27,28 Recently, Lajeunesse et al.23 measured asuite of antidepressants in treated biosolids from five differentCanadian sewage treatment plants, and the detected levels werein the nanograms per gram range (dry weight); the highestmean concentration was observed for citalopram at 1033 ng/g.The aim of this study was to develop a reliable and

sustainable, yet robust and quantitative, extraction method for asuite of commonly prescribed antidepressant pharmaceuticalsand selected degradates in biosolids. The procedure includesmicrosolvent extraction, followed by liquid chromatography/tandem mass spectrometry analysis. Extraction is often themost time-consuming step in preparation and analysis of solidand semisolid environmental samples, such as biosolids andsediments. With many extraction methods involving multiplepreparatory steps, hazardous byproducts and other wasteproducts may be produced. By elimination of unnecessarysteps, waste, energy use, and resources are reduced, whichtranslates into less solvents, chemicals, prep time, and costs.29

Microsolvent extraction was explored as a sustainable extractiontechnique due to its small sample volumes and solvent usage.

Figure 1. Chemical structures of antidepressants and degradation products, including isotopically labeled surrogate and internal standard.

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The target antidepressants in this study were the commonlyprescribed SSRIs and selective serotonin and norepinephrinereuptake inhibitors (SSNRIs) and included fluoxetine, sertra-line, paroxetine, citalopram, venlafaxine, and duloxetine (Figure1). The target degradates include norfluoxetine (or desmethyl-fluoxetine), norsertraline (or desmethylsertraline), and norven-lafaxine (or desmethylvenlafaxine). It is imperative to measurenot only “parent” pharmaceuticals but also their degradationproducts, as they can be as pharmacologically active as theparent compound.30 For example, norvenlafaxine is not onlythe degradation product of venlafaxine but also the activeingredient in Pristiq, a current prescription antidepressant.31

The validated methodology was then applied to biosolids, ormore specifically lagoon biosolid samples, destined for landapplication. Long-term effects of the land application ofbiosolids on the environment have not been studiedsufficiently; therefore investigation into this area is necessaryto fully understand the potential risks.11,22,25,27,32,33

■ EXPERIMENTAL SECTIONStandards and Reagents. All native and isotopically

labeled antidepressant solid standards (>98% purity) werepurchased from Toronto Research Chemical Inc. (North Fork,ON, Canada). HPLC-grade methanol and acetonitrile wereobtained from Pharmoco-Aaper (Brookfield, CT). Formic acid(88%) was obtained from Fisher Scientific (Fairlawn, NJ). ANanopure Diamond water purification system (VWR Interna-tional, West Chester, PA) dispensed ultrapure water. Individualstock solutions of the native and isotopically labeledantidepressant compounds were prepared in MeOH. Allstock solutions were stored in amber bottles in a freezer at−20 °C.Glassware Cleaning. All glassware was cleaned according

to guidelines from the U.S. Geological Survey.34 Briefly, theglassware was soaked (>1 h) in 1 g of Alconox/L of hot tapwater. Tap water was used to rinse the glassware until no soapresidue was visible. The glassware was then rinsed three timeswith ultrapure water and three times with the solvent of use.Environmental Sample Collection. Biosolids samples, or

more specifically lagoon biosolid samples, were collected at theWater Pollution Control Plant (WPCP) in Wooster, OH. Thisplant serves a community of approximately 26 000 people andtreats an average wastewater flow of 17 million L/day.35 Theplant uses activated aerobic sludge as the secondary treatmentand anaerobic digestion. UV disinfection has recently beeninstalled for tertiary treatment. In 2011 the plant removed 2.7million kg of suspended solids from wastewater.35 Afterundergoing treatment in the activated aerobic sludge (>20 h)and anaerobic digestion for approximately 20 days, thebiosolids are then added to the lagoon. The lagoon is a storagebasin for the digested biosolids until land application can occur,which is dependent upon weather, crop rotation, and harvestingcycles. No additional treatment process that is controlled by theplant occurs in the lagoon; however, additional treatment mayoccur through photolysis and/or by microbial degradation bybacteria present in the lagoon. The WPCP adds digestedbiosolids to the lagoon almost every day, about 190 000 L, anddecants excess water back to the headworks. A contract haulermixes the lagoon biosolids with residual lime and soda ash toensure that the total solids are at least 6% before landapplication.35 The biosolids are directly taken from the lagoonthroughout the year and are applied to local agricultural fieldsand in two nearby counties.35 More than 53 million L of

biosolids were taken from the lagoons in 2011, with themajority applied to land.35

Lagoon biosolid samples were collected in triplicate eachmonth from June 2012 to February 2013. A plastic containerwas used to scoop the biosolids from the lagoon, and it wasimmediately transferred to a 1 L amber jar. The amber jarscontaining the lagoon biosolids were stored in a refrigerator at4 °C until extraction, which was performed within 48 h ofcollection. The percentage of solids present in the collectedlagoon biosolids ranged from 3.5% to 7.4%, with an average of6.2%.

Microsolvent Extraction. Within 48 h of collection, thelagoon biosolids were extracted by microsolvent extractionmethod in triplicate. The 1 L amber jars were inverted 10 timesin order to thoroughly mix the lagoon biosolid samples.Samples (5 g) were withdrawn from the jars and added toindividual 15 mL BD Falcon tubes. Fluoxetine-d5 surrogate wasspiked into the sample tubes to a final concentration of 280 ng/kg. After >2 h, 5 mL of acetonitrile containing 0.1% formic acidwas added to the sample tubes. The samples were homogenizedfor 3 min (50% pulsing, 30% power) with a 3000 seriesultrasonic homogenizer (Biologics, Inc., Manassas, VA). Thenthe samples were sonicated for 30 min. After sonication, thesamples were centrifuged for 10 min at 10 000 rpm. Thesupernatant was transferred to a clean 15 mL Falcon tube andevaporated to dryness under a gentle stream of filtered air whileheating at 60 °C using a N-Evap 111 (OrganomationAssociates, Inc., Berlin, MA). After complete sample evapo-ration, the samples were reconstituted to 1 mL with methanol/water (50/50 v/v). The samples were vortexed for 2 min andsonicated again for 1 h. The 1 mL samples were transferred toamber HPLC vials and analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). After LC/MS/MSanalysis, samples were archived in a freezer at −20 °C.

Spike and Recovery. Spike and recovery experiments wereperformed at two concentrations to determine the accuracy andprecision of the microsolvent extraction method. The low andhigh sets were spiked to final concentrations of 10 and 280 ng/kg (wet weight), respectively, of individual antidepressants anddegradates. If present, the endogenous antidepressant concen-trations in the lagoon biosolids were subtracted from the totalquantified concentrations in the biosolids before the percentrecoveries of each analyte were calculated.

Liquid Chromatography/Tandem Mass Spectrometry.An Agilent 1200 series HPLC equipped with an Eclipse XDBC18 column (3.5 μm particle size, 2.1 × 150 mm) was used forseparation of the lagoon biosolid samples. The autosamplerinjected 25 μL of sample and was set to a 30 s needle wash withacetonitrile (ACN) containing 0.1% formic acid (FA). The LCsolvents were ultrapure water (solvent A) and acetonitrile(solvent B), both containing 0.1% formic acid. The LC gradientwas 10 min, plus a 4 min re-equilibration time, with the flowrate set to 0.25 mL/min. The gradient was as follows:

time (min) % ACN with 0.1% FA0 03 03.1 454 456 857 857.1 9510 9510.1 0

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The Agilent triple quadrupole 6410 mass spectrometer wasoperated in positive electrospray ionization (ESI) mode withthe gas temperature and flow set to 350 °C and 8 L/min,respectively. The nebulizer pressure was operated at 35 psi, thespray needle was held at 0 V, and the spray chamber was held at−4500 V. The mass spectrometer was operated in multiple-reaction-monitoring (MRM) mode, which scanned for bothprimary and secondary product ions of the precursor ion ofeach target analyte (Table 1).

Quantitation and Confirmation. After LC/MS/MSanalysis, all data were processed with MassHunter Quantitativesoftware. Calibration curves with concentrations ranging from 1to 1000 ng/kg were used for quantification of the targetantidepressants and fluoxetine-d5 in the samples. Thequantitative software used the weighted (1/x) linear least-squares regression model to generate trend lines. This modelminimizes the influence of lower concentration calibrationstandards in order to decrease the effect of measurementuncertainties on the correlation coefficient. All calibrationcurves contained a minimum of six data points and were notforced through 0. All correlation coefficients were high (R2 ≥0.99). The calibration curves were analyzed with each samplebatch run on the LC/MS/MS.All concentrations of antidepressants in the lagoon biosolids

were reported as wet weights. The reason for choosing wetweight over the more commonly used dry weight is 2-fold.First, the biosolids were extracted as collected, which were“wet” samples, approximately 6% solids. To report a true dryweight of the analyte in the biosolids, the sample must be driedprior to sample preparation. Second, the contract hauler doesnot further dry the lagoon biosolids before their landapplication. Therefore, by measuring the antidepressants anddegradates in the biosolids as they are applied (∼6% solids), abetter representation is provided of their occurrence and levelsbeing introduced to terrestrial environments in both theadsorbed and dissolved phases of biosolids.To determine the limit of detection (LOD) and limit of

quantitation (LOQ) of each antidepressant and degradationproduct, the average noise level for each analyte wasdetermined from the chromatograms within the biosolidmatrices. The average noise signals were converted toconcentration values (nanograms per kilogram) through

extrapolation of the trendline equation of each analytes’respective calibration curve. The noise concentrations weremultiplied by 3 to determine the LOD and by 10 for the LOQ.Any analyte detected above the LOD but below the LOQ wasreported as ≤LOQ.

■ RESULTS AND DISCUSSIONBiosolid Sample Preparation Optimization. Initial

sample preparation of the lagoon biosolid samples for thisstudy utilized the method of accelerated solvent extraction (alsoreferred to as pressurized liquid extraction). The antidepres-sants were extracted by accelerated solvent extraction using amethod described elsewhere for pharmaceuticals and personalcare products, which included the antidepressant fluoxetine,25

and is briefly described in the Supporting Information. Due tothe poor extraction recoveries and the large amount of solventaccrued, other extraction techniques were explored. Microscalesolvent extraction was a desirable alternative to acceleratedsolvent extraction for this study. It is also a green technique as itemploys small sample volumes and solvent usage, therebyreducing generated waste and time-consuming preparatorysteps. Microscale solvent extraction has been used for a widevariety of target chemicals; a brief literature search reports thatmicroscale solvent extraction has been used for pesticides,36

perchlorate,37 and lipids.38

Since microscale solvent extraction has few preparatory steps,there are few parameters to optimize. For this study, 1 and 5 gsample sizes were compared. The target analytes weresufficiently detected in the lagoon biosolids at both volumes;however, the 5 g sample volume was chosen as it provided alarger signal-to-noise ratio for the antidepressants as comparedto the 1 g sample volume. The solvents methanol andacetonitrile both produced similar extraction efficiencies;however, acetonitrile was chosen as the extraction solvent asit had better reproducibility. Acidification was previouslydemonstrated to to increase recovery efficiency in antidepres-sants;19 therefore, 0.1% formic acid was added to theacetonitrile.The accuracy and precision of the extraction method was

determined from spike and recovery experiments performed athigh (280 ng/kg) and low (10 ng/kg) concentrations in theWPCP lagoon biosolids. Recoveries for all analytes typicallywere good with an average of 96% for the high and low sets;however, some notable outliers were observed, leading to arange of 12−160% (Table 2). The lower observed recoveries ofsertraline and norsertraline may be explained by their highsorption capacities to sludge.23 The recoveries from thismicrosolvent extraction method are comparable to previouslyreported values in the literature for methods that include moreinvolved sample preparations of antidepressants from biosolids.For example, fluoxetine and sertraline have 100% and 74%recoveries, respectively, with EPA Method 1694, which issequential liquid−liquid extractions followed by solid-phaseextraction.26,28 Also, Lajeunesse et al.23 reported recoveries of44−101% for antidepressants and degradates using minimalsample preparation followed by solid-phase extraction, andRadjenovic et al.39 reported recoveries of 16% and 59% forfluoxetine and paroxetine, respectively, in sludge samples usingaccelerated solvent extraction followed by solid-phase extrac-tion.The limit of detection (LOD) and limit of quantitation

(LOQ) of the microsolvent extraction method weredetermined for each antidepressant and degradation product

Table 1. Mass Spectrometer Parameters and Ion TransitionsUsed for Identification and Quantitation of Antidepressantsand Their Degradation Productsa

primary secondary

compdprecursorion, m/z

production, m/z

collisionenergy,eV

production, m/z

collisionenergy,eV

fluoxetine-d5b 315 44 15 148 15

fluoxetine 310 44 10 148 15sertraline 306 159 15 275 10venlafaxine 278 260 10 121 10citalopram 325 262 15 109 15paroxetine 330 192 15 70 15duloxetine 298 154 15 44 15norfluoxetine 296 30 15 134 15norsertraline 292 275 15 159 15norvenlafaxine 264 246 15 58 15

aFragmentor voltage was 60 V for all compounds. bSurrogate.

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in lagoon biosolids. LOD and LOQ were defined as theconcentration of each antidepressant analyte that produced asignal-to-noise ratio of 3:1 and 10:1, respectively, in the lagoonbiosolid samples. The LOD values were determined to rangefrom 0.36 (citalopram) to 8.0 (norsertraline) ng/kg wet weight(Table 2). The individual LOQs ranged from 1.2 (citalopram)to 27 (norsertraline) ng/kg wet weight.Liquid Chromatgraphy/Tandem Mass Spectrometry.

Chromatographic separation of the detected target antidepres-sants and degradation products was achieved for the calibrationstandards (not shown) and for the lagoon biosolid samples(Figure 2). The ratio of the two product ions (quantitation andconfirmation ions) from fragmentation of the target analyte(precursor ion) in combination with the retention time wasused to identify the target antidepressants. The quantitation ionwas defined as the most abundant product ion for each of theanalytes, and its concentration was reported for the targetantidepressants (Table 3).Application to Environmental Samples. The range of

concentrations of detected target antidepressants varied from8.5 ± 0.7 (norfluoxetine) to 420 ± 100 (sertraline) ng/kg wetweight in the lagoon biosolid samples (Table 3). Of the sixantidepressants and three degradates that were detected,sertraline and its primary degradation product, norsertraline,were the most abundant analytes measured. Fluoxetine,citalopram, paroxetine, norfluoxetine, and norvenlafaxine werealso detected every month in addition to sertraline andnorsertraline. A comparison of the antidepressants present inthe WPCP lagoon biosolids to those previously reported inbiosolids show similar antidepressant profiles. Sertraline andnorsertraline were the first and second most abundantantidepressants observed in a recent study that looked for sixantidepressants in biosolids collected from anaerobic digestiontanks from a wastewater treatment plant in Ithaca, NY.33 Thedetected concentrations for sertraline and norsertraline were104 and 93 ng/g (dry weight), respectively.33 Sertraline wasalso the most abundant antidepressant measured in thearchived composite biosolids from the 2001 EPA NationalSewage Sludge Survey.28 The prevalence of sertraline and itsprimary degradation product in sludge may be explained, inpart, by their high sorption capacities to wastewater solids.22,23

In the study of Lajeunesse et al.,23 sertraline was determined to

have the highest sorption coefficient (log Kd = 4.5) ascompared to the other SSRIs and SSNRIs.Venlafaxine and duloxetine were not always detected above

the LOQ. Fluvoxamine was never detected in the lagoonbiosolid samples and thus was not included in the methodvalidation. This could be due to the values of sorptioncapacities for these antidepressants. Kwon and Armbrust22

found that fluvoxamine had the lowest sorption capacity of theSSRI antidepressants. Lajeunesse et al.23 and Horsing et al.40

determined that the sorption coefficient for venlafaxine was 2orders of magnitude smaller than that for sertraline. Analternative explanation for the low observed levels could be thatthese antidepressants are more susceptible to degradation. Inthe case of venlafaxine, its degradation product norvenlafaxinewas observed and measured in every monthly sample. With theexception of the September sample, norvenlafaxine wasdetected at higher concentrations than venlafaxine (Table 3and Figure 3). The higher observed concentrations fornorvenlafaxine as compared to venlafaxine could be explainedby its use as the active ingredient in the current antidepressantPristiq.31 Alternatively, the higher abundance of norvenlafaxinecould be explained by the degradation of venlafaxine. Inmicrocosm studies conducted with aerobic wastewater sludgecollected from the WPCP plant, venlafaxine was shown tosignificantly degrade after 2 days of treatment (unpublisheddata).The variable concentrations and large standard deviations of

the antidepressants detected in lagoon biosolid samplescollected monthly between June 2012 and February 2013were expected due to the heterogeneous nature of the biosolids(Table 3 and Figure 3). There is not a standard residence timefor the biosolids in the lagoons. Until the lagoon biosolids aretaken for land application, they remain in the lagoon and mixwith the constantly inflowing biosolids, which enter primarilyfrom the anaerobic digestors.35 Other contributing factors tothe heterogeneous character of the lagoon biosolids include theplant residual lime and soda ash, which is added at nonuniformrates and locations, and the fact that lagoons are open air andsusceptible to dilution from precipitation events. These factorsimpact the concentrations of the individual antidepressantsdetected in the monthly lagoon biosolid samples. A suite ofantidepressants was consistently detected in the lagoon biosolidsamples, and thus, antidepressants are being introduced toterrestrial environments through the land application of thesebiosolids.Temperature has also been found to impact the survival of

antidepressants during wastewater treatment. Lajeunesse et al.23

discovered that the efficiency of antidepressant degradation bymicroorganisms decreased at low temperatures. In a wastewatertreatment plant in Canada, bioremediation was found to be11% less efficient for removal of antidepressants at 11.3 °C(April) than at 21.2 °C (September).23 Seasonal variationbetween sampling therefore could impact the concentrations ofantidepressants in the lagoon biosolids, where high temper-atures correspond to reduced antidepressant concentrations.However, this trend was not observed in this study, where thehighest concentrations of antidepressants in the biosolids werefrom samples collected in December, one of the colder months,when the average high temperature in northeast Ohio is 3 °C(Table 3 and Figure 3). Overall, there were no observedseasonal trends for the occurrence of antidepressants inbiosolids.

Table 2. Mean Percent Recoveries Obtained after Extraction,and Limits of Quantitation and Detection, of IndividualAntidepressants and Degradates in Biosolids

mean recovery (% ± SD)

compd low spikea high spikeaLOQ (ng/

kg)LOD (ng/

kg)

fluoxetine-d5b nac 70 ± 10 na na

fluoxetine 74.8 ± 0.9 102 ± 4 18 0.66sertraline 58 ± 6 62 ± 6 15 3.0venlafaxine 88.7 ± 0.3 108 ± 6 9.1 2.7citalopram 30.7 ± 0.8 105 ± 5 1.2 0.36paroxetine 127.7 ± 0.4 142.5 ± 0.4 7.0 2.1duloxetine 106.4 ± 0.5 130 ± 1 6.3 1.9norfluoxetine 120 ± 1 118 ± 2 6.0 1.8norsertraline 12 ± 1 74 ± 1 27 8.0norvenlafaxine 137 ± 3 160 ± 20 11 3.3

aLow and high sets were spiked to final concentrations of 10 and 280ng/kg (wet weight), respectively, of individual antidepressants anddegradates. bSurrogate. cna = not analyzed.

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Figure 2. Representative LC/MS/MS chromatogram of antidepressants and degradates determined in lagoon biosolids (August 2012 sample). Foreach target antidepressant and degradate, two precursor-to-product ion transitions were monitored, a quantitative and a qualitative transition, toincrease specificity. Both monitored ion transitions are shown in the representative chromatogram for each antidepressant or degradate. A box isused to identify peaks that correspond to the target analyte, according to retention times determined by reference standards.

Table 3. Mean Concentrations for Antidepressants and Degradation Products in Monthly Lagoon Biosolid Samples Collectedfrom June 2012 to February 2013

mean concn (n = 3), ng/kg wet weight ± SD

overall mean June July Aug Sept Oct Nov Dec Jan Feb

sertraline 230 ± 40 120 ± 13 260 ± 60 170 ± 10 310 ± 60 240 ± 30 170 ± 30 420 ± 100 140 ± 20 220 ± 30fluoxetine 60 ± 10 70 ± 50 65 ± 9 51 ± 9 60 ± 10 42 ± 5 38 ± 6 90 ± 20 35 ± 3 54 ± 5venlafaxine 75 ± 40 <LOQ 16 ± 5 <LOQ 260 ± 90 10 ± 1 <LOQ <LOQ <LOQ 14 ± 3citalopram 110 ± 20 39 ± 5 160 ± 50 94 ± 4 150 ± 20 95 ± 8 70 ± 10 250 ± 40 69 ± 5 80 ± 10paroxetine 34 ± 2 17 ± 2 56 ± 4 31 ± 1 44 ± 8 23 ± 3 28 ± 2 60 ± 7 18 ± 4 30 ± 3duloxetine 25 ± 4 <LOQ <LOQ 30 ± 1 32 ± 6 10 ± 3 12 ± 3 40 ± 10 19.2 ± 0.6 30 ± 2norsertraline 200 ± 30 56 ± 3 250 ± 70 110 ± 5 180 ± 30 310 ± 50 260 ± 30 370 ± 70 120 ± 10 180 ± 20norfluoxetine 45 ± 3 8.5 ± 0.7 42 ± 7 32 ± 4 50 ± 10 49 ± 4 54 ± 3 90 ± 10 36 ± 3 42 ± 4norvenlafaxine 70 ± 10 26 ± 7 23.6 ± 0.3 60 ± 5 87 ± 6 58 ± 5 80 ± 9 150 ± 30 60 ± 20 100 ± 20

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The results of this study confirm that antidepressants andtheir degradates are being introduced to terrestrial environ-ments through the land application of biosolids. In theseenvironmental samples, typical concentrations of the anti-depressants were in the nanograms per kilogram (wet weight)range. Further research is needed to determine the occurrence,distribution, and fate of antidepressants in the biosolid-amended soils. To the best of our knowledge, this study isthe first to report the consistent detection of a suite ofantidepressants in biosolids destined for land application over amultimonth study.

■ ASSOCIATED CONTENT

*S Supporting InformationAdditional text with details on accelerated solvent extraction.This material is available free of charge via the Internet athttp://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*Phone 330-263-2645; fax 330-263-2386; e-mail mschultz@wooster.edu.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This research was supported in part by grants from theNational Science Foundation (CBET-1235900 and CHE-0821110). Additional support was provided by a grant toThe College of Wooster from the Howard Hughes MedicalInstitute through the Undergraduate Science EducationProgram. We also thank Dr. Shelly Hoffman for her guidanceand assistance. Our gratitude is extended to Kevin Givens, LeeTroyer, and Otis of the WPCP for assistance in the monthlysample collection.

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Figure 3. Mean concentration (±standard deviation, SD) of the antidepressants and degradation products detected in the lagoon biosolid samplescollected between June 2012 and February 2013.

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