www.elsevier.com/locate/chemosphere

Chemosphere 65 (2006) 1990–1998

Broad range analysis of endocrine disruptors and pharmaceuticalsusing gas chromatography and liquid chromatography tandem

mass spectrometry

Rebecca A. Trenholm *, Brett J. Vanderford, Janie C. Holady,David J. Rexing, Shane A. Snyder

Water Quality Research and Development Department, Southern Nevada Water Authority, 1350 Richard Bunker Avenue,

Henderson, NV 89015, United States

Received 27 February 2006; received in revised form 28 June 2006; accepted 3 July 2006Available online 1 September 2006

Abstract

Endocrine disrupting compounds (EDCs) and pharmaceuticals and personal care products (PPCPs) have been globally detected inimpacted natural waters. The detection of trace quantities of EDCs and PPCPs in the environment is of great concern since some ofthese compounds have known physiological responses at low concentrations. EDCs can have a wide range of polarities, acidic and basicmoieties, and exist in trace quantities, which often requires numerous complex extractions, large sample collection volumes, and multipleinstrumental analyses. A comprehensive method has been developed allowing for the analysis of 58 potential EDCs in various watermatrices using a single solid-phase extraction (SPE) of a 1 L sample with subsequent analyses using both gas chromatography and liquidchromatography, each coupled with tandem mass spectrometry (GC–MS/MS and LC–MS/MS). Instrument detection limits rangedbetween 0.12–7.5 pg with corresponding method reporting limits of 1–10 ng l�1 in water. Recoveries for most compounds were between50% and 112% with good reproducibility (RSD 6–22%).� 2006 Elsevier Ltd. All rights reserved.

Keywords: Endocrine disruptor; Solid-phase extraction; GC–MS/MS; LC–MS/MS

1. Introduction

Research of compounds that can mimic the natural hor-mones of animals, referred to as endocrine disrupting com-pounds (EDCs), has been reported for over 70 years andhas recently been linked to physiological effects in animals(Snyder et al., 2003). Likewise, several pharmaceuticalshave been ubiquitously detected in wastewater effluentsfrom various locations around the world (Halling-Sorensenet al., 1998; Daughton and Ternes, 1999; Ternes et al.,2001; Miao et al., 2002; Bruchet et al., 2002; Kolpinet al., 2002; Vanderford et al., 2003; Anderson et al., 2005).

0045-6535/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.chemosphere.2006.07.004

* Corresponding author. Tel.: +1 702 856 3658; fax: +1 702 856 3647.E-mail address: beck.trenholm@snwa.com (R.A. Trenholm).

The continued improvements in analytical methods haveallowed researchers to investigate the fate and transport ofEDCs, with particular attention focused on the occurrenceof natural and synthetic steroids in the aquatic envi-ronment as a result of municipal wastewater discharge(Stumm-Zollinger and Fair, 1965; Tabak et al., 1981;Ternes et al., 1999a,b; Huang and Sedlak, 2001; Lee andLiu, 2002; D’Ascenzo et al., 2003). Trace concentrationsof steroids have been shown to induce physiologicalresponses in organisms exposed to wastewater effluents(Jobling et al., 1998; Kramer et al., 1998; Routledgeet al., 1998; Snyder et al., 2004; Brennen et al., 2006; Hoek-stra et al., 2006). Many surface waters that provide rawdrinking water are impacted by WWTP effluent, thus rais-ing public concern over possible human health impactsfrom contaminated drinking water. Therefore, information

Table 1Target compounds, use, molecular weight (MW), pKa, water solubility and log Kow

Compound Use MW pKa Water solubility (mg l�1) LogKow

GC–MS/MS

Acenaphthylene PAH 152.2 na 16.1a 3.94a

Aldrin Pesticide 364.9 na 0.017a 6.50l

Alpha-BHC Pesticide 290.8 na 2a 3.80l

Alpha-chlordane Pesticide 409.8 na 0.056a 5.80l

Anthracene PAH 178.2 na 0.0434a 4.45l

Benz[a]anthracene PAH 228.3 na 0.0094a 5.79l

Benzo[a]pyrene PAH 252.3 na 0.00162a 6.13a

Benzo[b]fluoranthene PAH 252.3 na 0.0015a 5.78a

Benzo[k]fluoranthene PAH 252.3 na 0.00055d 6.64d

Beta-BHC Pesticide 290.8 na 0.24a 3.78l

Chrysene PAH 228.3 na 0.002a 5.81a

DDD Pesticide 320.0 na 0.09a 6.02a

DDE Pesticide 318.0 na 0.04a 6.96l

DDT Pesticide 354.5 na 0.0055a 6.91l

Delta-BHC Pesticide 290.8 na 10a 4.14a

Dieldrin Pesticide 380.9 na 0.195a 5.20l

Endrin Pesticide 380.9 na 0.25a 5.20a

Fluoranthene PAH 202.3 na 0.26a 5.16l

Fluorene PAH 166.2 na 1.89a 4.18l

Galaxolide Personal care product 258.4 na 1.75f 5.30f

Gamma-BHC Pesticide 290.8 na 7.3a 3.76l

Gamma-chlordane Pesticide 409.8 na 0.0001g 6.26c

Heptachlor Pesticide 373.3 na 0.18a 6.10a

Heptachlor epoxide Pesticide 389.3 na 0.2a 4.98a

Methoxychlor Pesticide 345.7 na 0.1a 5.08l

Metolachlor Pesticide 283.8 na 530a 3.13l

Mirex Pesticide 545.5 na 0.085a 6.89a

Musk ketone Personal care product 294.3 na 0.46f 4.30f

Naphthalene PAH 128.2 na 31a 3.30l

Phenanthrene PAH 178.2 na 1.15a 4.56l

Pyrene PAH 202.3 na 0.135a 4.88l

LC–MS/MS

Acetaminophen Pharmaceutical 151.2 9.38a 1.4E+4a 0.46a

Androstenedione Steroid 286.4 na 57.8a 2.75a

Atrazine Pesticide 215.7 1.7a 34.7a 2.61a

Caffeine Personal care product 194.2 10.4a 2.16E+4a �0.07a

Carbamazepine Pharmaceutical 236.3 13.9e 17.7a 2.45a

DEET Personal care product 191.3 na 912a 2.18a

Diazepam Pharmaceutical 284.7 3.4a 50a 2.82a

Diclofenac Pharmaceutical 318.1 4.15a 2.37a 4.51a

Dilantin Pharmaceutical 252.3 8.33a 32a 2.47a

Erythromycin Pharmaceutical 733.9 8.88a 1.44a 3.06a

Estradiol Steroid 272.4 10.4k 3.6a 4.01a

Estriol Steroid 288.4 10.4k 441a 2.45a

Estrone Steroid 270.4 10.4 j 30a 3.13a

Ethynylestradiol Steroid 296.4 10.4k 11.3a 3.67a

Fluoxetine Pharmaceutical 309.3 9.5b 60.3a 4.05a

Gemfibrozil Pharmaceutical 250.3 na 19h 4.77a

Hydrocodone Pharmaceutical 299.4 8.48e 6870h 2.16a

Ibuprofen Pharmaceutical 206.3 4.91a 21a 3.97a

Iopromide Pharmaceutical 791.1 na 23.8a �2.05a

Meprobamate Pharmaceutical 218.3 na 4700a 0.70a

Naproxen Pharmaceutical 230.3 4.15a 15.9a 3.18a

Oxybenzone Personal care product 228.2 na 68.6a 3.79a

Pentoxifylline Pharmaceutical 278.3 na 7.7E+4a 0.29a

Progesterone Steroid 314.5 na 8.81a 3.87a

Sulfamethoxazole Pharmaceutical 253.3 6.0h 610a 0.89a

Testosterone Steroid 288.4 na 23.4a 3.32a

Triclosan Personal care product 289.5 8.14i 10a 4.76a

(continued on next page)

R.A. Trenholm et al. / Chemosphere 65 (2006) 1990–1998 1991

Table 1 (continued)

Compound Use MW pKa Water solubility (mg l�1) LogKow

Trimethoprim Pharmaceutical 290.3 7.12a 400a 0.91a

TCEP Personal care product 285.5 na 7000a 1.44a

a Environmental Science Database SRC (2005) PhysProp.b Hendrick (2000).c Environmental Science Center Interkow (calculated).d PA Department of Environmental Protection (2005).e ROHM and HAAS (2005).f Ospar Commission (2004).g EPA (2005) technical fact sheet.h Glaxosmithkline UK.i Danish Environmental Protections Agency (2003).j Nghiem et al. (2004).

k Deborde et al. (2005).l Sabljic et al. (1995).

1992 R.A. Trenholm et al. / Chemosphere 65 (2006) 1990–1998

is needed on the removal of EDCs and pharmaceuticals bydrinking and wastewater treatment processes.

EDCs include a broad spectrum of compounds withlarge differences in polarities (log Kow’s), neutral, acidic,or basic moieties, and molecular weights thus makingthe development of analytical techniques quite challeng-ing. There are many analytical methods available forextraction and detection of certain EDCs from variousmatrices (Petrovic et al., 2002; Benijts et al., 2004; Lagunaet al., 2004; Richarson, 2004). However, most requiresome or all of the following: multiple extractions, largesample volumes, derivatization and extensive sample cleanup. Some methods can only detect a small range of similarcompounds (i.e., estrogen steroids). This paper discussesthe development of a comprehensive method for the anal-yses of several potential EDCs and pharmaceuticals invarious water matrices. Using sample volumes of 100 mlto 1 L and a single solid-phase extraction (SPE), 58 com-pounds were analyzed using GC–MS/MS and LC–MS/MS. The target compounds were chosen to represent awide variety of compound classes, as part of a larger pro-ject determining contaminant removal during water treat-ment processes (AwwaRF Project #2758), based onoccurrence and their physico-chemical properties. Com-pounds selected have logKow’s ranging from �2.05 to6.91, molecular weights from 128 to 791, and include somewith acidic or basic functional groups (Table 1). The tar-get compounds include pharmaceuticals, polycyclic aro-matic hydrocarbons (PAHs), pesticides, natural andsynthetic steroids, and personal care products. Althoughprevious methods exists for some, this is the first time,to the knowledge of the authors, that a method has beendeveloped which can test a wide variety of compoundswith a single extraction in various water matrices withng l�1 reporting limits using tandem mass spectrometrytechniques. This method was applied to a variety of watermatrices to determine robustness and to determine theoccurrence and concentration of these target compoundsin wastewater influent and effluent, ground, surface anddrinking water.

2. Experimental

2.1. Standards and reagents

All standards and reagents used were of the highest pur-ity commercially available. All standards were obtainedfrom Sigma–Aldrich (St. Louis, MO) except polyaromatichydrocarbons and pesticides from Ultra Scientific (NorthKingstown, RI); mirex, atrazine, and DEET from Accu-standard (New Haven, CT); fluoxetine and iopromide fromthe United States Pharmacopeia (Rockville, MD); hydro-codone from Cerilliant (Round Rock, Texas); [13C10]-trans-chlordane, [13C10]-heptachlor, [13C6]-alpha-BHC,[13C12]-DDE, [13C6]-metolachlor, [13C12]-methoxychlor,[13C3]-caffeine, [13C3]-atrazine, [13C2]-estradiol, diazepam-d5, DDD-d8, anthracene-d10, acenaphthylene-d8, fluorene-d10, benzo[a]pyrene-d10 and perylene-d12 from CambridgeIsotope Laboratories (Andover, MA). All solvents weretrace analysis grade from Burdick and Jackson (Muskegon,MI) except formic acid and methyl-t-butyl ether (MTBE)from EM Science (Gibbstown, NJ).

2.2. Sample collection and preservation

Samples were collected in 1 l ultra clean, pre-silanized,amber glass bottles (Eagle–Picher, Miami, OK) as sug-gested by Ahrer et al. (2000), to prevent compounds frombinding to the glass surface of the collection bottles. Sam-ple bottles were kept cold on ice while transporting back tothe laboratory. Once received, samples were immediatelypreserved by pH adjustment (pH 2) using sulfuric acidand stored at 4 �C until extraction. All samples wereextracted within 14 days of collection. Wastewater influentand effluent samples were filtered prior to extraction using90 mm GF/F filters (Whatman, England).

2.3. Solid-phase extraction

Samples were extracted in batches of six using 500 mghydrophilic–lipophilic balance (HLB) cartridges from

Sample acidified / filtered Surrogate standards added

Solid Phase Extraction of 1 Liter sample

Fraction 1 → 1 ml MeOH / MTBE

500 μl split

LLE → 500 μl

Internal standards added

APCI Positive

LC-MS/MS analysis

GC-MS/MS analysis

EI Positive

500 μl split

Fraction 2 → 3 mlDCM

ESI Negative

ESI Positive

Fig. 1. Flow chart of sample extraction process and analysis.

R.A. Trenholm et al. / Chemosphere 65 (2006) 1990–1998 1993

Waters Corp. (Milliford, MA). All extractions were per-formed using an Autotrace automated SPE system(Zymark Corp., Hopkington, MA). The SPE cartridgeswere conditioned with 5 ml dichloromethane (DCM),5 ml tert-butyl methyl ether (MTBE), 5 ml methanol, and5 ml reagent water (Barnstead Nanopure). One-liter sam-ples were spiked with 40 ll of a 2.5 mg l�1 solution ofGC–MS/MS surrogate standards ([13C12]-DDE, [13C10]-trans-chlordane, [13C10]-heptachlor, anthracene-d10, fluo-rene-d10, perylene-d12) and 20 ll of a 2.5 mg l�1 solutionof LC–MS/MS surrogate standards (diazepam-d5, testos-terone-d3, [13C3]-caffeine, [13C3]-atrazine, and [13C2]-estra-diol) and thoroughly mixed. Surrogates were spikedpost-filtration in wastewater samples. The samples werethen loaded onto SPE cartridges at 15 ml min�1. Aftersample loading, the SPE cartridges were rinsed with 5 mlreagent water and dried for 60 min with nitrogen. TheSPE cartridges were eluted in two separate fractions. Thefirst fraction (F1), which was used for LC–MS/MS andGC–MS/MS analysis, was eluted with 5 ml of methanolfollowed by 5 ml of 10/90 (v/v) methanol/MTBE. Theeluted extract was collected in a 15 ml calibrated glass vial(Zymark Corp., Hopkington, MA). The second fraction(F2) was eluted using 10 ml DCM into a separate 15 mlvial. F1 was concentrated with a gentle stream of nitrogento a final volume of 1 ml. Five hundred ll was removedfrom F1 and placed into a 2 ml autosampler vial for LC/MS/MS analysis, while the remaining 500 ll was retainedfor liquid/liquid extraction. F2 was concentrated to 3 mlwith a gentle stream of nitrogen.

2.4. Liquid/liquid extraction

Prior to GC–MS/MS analysis, a liquid/liquid extraction(LLE) was performed on the remaining 500 ll from F1.First, 2.5 ml of a 25% sodium chloride (w/v) solution wasadded to F1 and vortexed. Then 3 ml of 10/90 (v/v)DCM/hexane solution was added to F1 and vortexed.The sample was allowed to settle, and the top layer(organic) removed and combined with F2. F1 wasextracted twice and the resulting extracts combined withF2. The final combined organic extracts were then concen-trated with a gentle stream of nitrogen to 1 ml. Two ml ofiso-octane were added and the extract was concentrated to�450 ll. Twenty-five ll of a 2 mg l�1 solution of internalstandards ([13C6]-alpha-BHC, [13C6]-metolachlor, ace-naphthylene-d8, benzo[a]pyrene-d10 and DDD-d8) wereadded. The extract was adjusted to a final volume of500 ll using iso-octane and transferred to a 2 ml autosam-pler vial for GC–MS/MS analysis. A method summary isillustrated in Fig. 1.

2.5. Gas chromatography

A Varian (Walnut Creek, CA) CP-3800 Gas Chromato-graph with a CP-8400 autosampler was used for all GC–MS/MS analyses. The injector (Varian 1177) was operated

in splitless mode with a SiltekTM deactivated glass liner withglass frit (Restek, Bellefonte, PA). Analytes were separatedon a 30 m · 0.25 mm ID · 0.25 lm DB5-MS column (J &W, Agilent, Palo Alto, CA) using a 1.0 ml min�1 heliumflow with an initial pressure pulse of 45 PSI for 0.85 min.The temperature program was as follows: 90 �C, hold for2.0 min; 90–150 �C @ 20 �C min�1; 150–280 �C @3 �C min�1, hold for 5.0 min; 280–315 �C @ 30 �C min�1,hold for 2.5 min. An injection volume of 2 ll was used forall analyses.

2.6. Liquid chromatography

The liquid chromatography methodology was publishedpreviously (Vanderford et al., 2003). Briefly, a binary pump(Agilent G1312A, Palo Alto, CA) and an autosampler(HTC-PAL, CTC Analytics, Zwingen, Switzerland) wereused for all analyses. All analytes were separated using a250 · 4.6 mm Synergi Max-RP C12 column with a 4 lmparticle size (Phenomenex, Torrance, CA). A binary gradi-ent consisting of 0.1% formic acid (v/v) in water (A) and100% methanol (B) at a flow rate of 700 ll min�1 was used.The gradient was as follows: 5% B held for 3.5 min,increased linearly to 80% by 10 min and held for 3 min,stepped to 100% and held for 8 min. A 9 min equilibrationstep at 5% B was used at the beginning of each run to bringthe total run time per sample to 30 min. An injection vol-ume of 10 ll was used for all analyses.

Table 2Instrument detection limit (IDL), method detection limits (MDL), reportinglimits (RL), percent recovery, and percent relative standard deviation (RSD)

Compound IDL(pg)

MDL(ng l�1)

RL(ng l�1)

Percentrecovery

PercentRSD

GC–MS/MS n = 10 n = 11 n = 21Acenaphthylene 4.5 2.38 10 69 20Aldrin 2.5 4.08 10 48 13Alpha-BHC 2.3 2.63 10 59 19Alpha-chlordane 2.2 3.57 10 64 13Anthracene 2.3 2.25 10 83 7Benz[a]anthracene 1.2 2.48 10 82 7Benzo[a]pyrene 4.3 2.35 10 67 9Benzo[b]fluoranthene 7.5 3.38 10 71 8Benzo[k]fluoranthene 2.5 7.84 10 69 8Beta-BHC 1.3 1.81 10 81 11Chrysene 2.7 3.83 10 77 7DDD 1.6 1.71 10 64 9DDE 3.8 2.32 10 65 18DDT 2.0 4.36 10 71 11Delta-BHC 3.9 4.41 10 74 12Dieldrin 4.5 4.68 10 66 22Endrin 3.9 3.24 10 82 21Fluoranthene 2.0 3.17 10 91 8Fluorene 2.6 1.99 10 71 8Galaxolide 1.2 5.75 10 30 12Gamma-BHC 1.8 3.04 10 65 17Gamma-chlordane 1.7 4.37 10 55 13Heptachlor 1.6 4.19 10 55 13Heptachlor Epoxide 3.6 4.63 10 74 13Methoxychlor 2.4 2.72 10 75 12Metolachlor 3.5 2.24 10 75 17Mirex 0.83 1.84 10 46 16Musk Ketone 6.7 5.50 10 72 16Naphthalene 1.5 3.76 10 56 22Phenanthrene 1.6 5.36 10 84 6Pyrene 1.6 1.12 10 92 8

LC–MS/MS n = 10 n = 12 n = 17Acetaminophen 0.30 0.139 1 41 14Androstenedione 0.64 0.210 1 96 12Atrazine 0.15 0.141 1 89 8Caffeine 0.28 1.13 10 93 10Carbamazepine 0.43 0.211 1 91 12DEET 0.24 0.442 1 81 19Diazepam 0.28 0.168 1 80 19Diclofenac 0.12 0.137 1 83 11Dilantin 0.94 0.332 1 86 11Erythromycin 0.17 0.221 1 71 10Estradiol 0.62 0.297 1 92 14Estriol na 0.587 1 101 9Estrone na 0.534 5 90 7Ethynylestradiol 0.87 0.256 1 92 11Fluoxetine 0.31 0.187 1 80 7Gemfibrozil 0.096 0.165 1 94 10Hydrocodone 0.23 0.293 1 112 7Ibuprofen 0.32 0.184 1 96 15Iopromide 0.72 0.577 1 91 9Meprobamate 0.68 0.159 1 96 11Naproxen 0.57 0.225 1 91 9Oxybenzone 0.35 0.479 1 68 19Pentoxifylline 0.28 0.225 1 84 15Progesterone 0.38 0.261 1 90 12Sulfamethoxazole 0.35 0.217 1 72 16TCEP 3.2 1.59 10 75 18Testosterone 0.41 0.126 1 83 9Triclosan 0.68 0.390 1 79 17Trimethoprim 0.30 0.194 1 91 9

na = not available.

1994 R.A. Trenholm et al. / Chemosphere 65 (2006) 1990–1998

2.7. Mass spectrometry

For analysis of GC–MS/MS compounds, mass spec-trometry was performed using a Varian 2200 ion trap massspectrometer (Walnut Creek, CA). All analyses were per-formed using multiple reaction monitoring (MRM) in posi-tive electron impact mode. For analysis of LC–MS/MScompounds, mass spectrometry was performed using anAPI 4000 triple quadrupole mass spectrometer (AppliedBiosystems, Foster City, CA) using MRM with electro-spray ionization (ESI) in both positive and negative modesand atmospheric pressure chemical ionization (APCI) inpositive mode. Full LC–MS/MS method parameters havebeen previously published (Vanderford et al., 2003). AllGC–MS/MS and LC–MS/MS ion transitions are providedin Supplementary material 1. An example chromatogramof GC–MS/MS analysis is shown in Supplementary mate-rial 2.

2.8. Recoveries and calibration

Instrument detection limits (IDL), method detectionlimits (MDL), method reporting limits (MRLs), percentrecovery and percent relative standard deviations are givenin Table 2. IDL studies were performed according to Vand-erford et al. (2003). GC–MS/MS compounds were adjustedaccordingly to their experimentally determined mass bal-ance correction factors, which range from 0.5 to 1.0 (Table3). Resulting compound recoveries and data were adjustedaccordingly.

MRLs were chosen based on a value of 3–5 times theMDL and set as the lowest calibration point, for a signalto noise ratio greater than 10 for all compounds. A fewcompounds had reporting limits less than three times theMDL, such as triclosan and oxybenzone, as these werecompounds in which a trace amount of blank contamina-tion was unavoidable.

For GC–MS/MS, analyte quantitation was performedusing internal standard calibration with linear or quadraticregression with 1/x2 weighting. LC–MS/MS quantitationwas performed using external standard calibration with lin-ear or quadratic regression with 1/x2 weighting. GC–MS/MS calibration points were at 10, 25, 50, 100, 250 and500 lg l�1, while LC–MS/MS calibration points were at1.0, 2.5, 5.0, 10, 25, 50, 100 and 200 lg l�1. Correlationcoefficients were consistently 0.990 or greater. Supplemen-tary material 3 lists GC–MS/MS compounds and theiroccurrence in 15 extracted unspiked, deionized watersamples.

Matrix spikes for GC–MS/MS compounds were ana-lyzed using surface water with wastewater effluent influencecollected from the Colorado River (Table 4). Matrix spikeresults for LC–MS/MS compounds can be found in Vand-erford et al. (2003). Evaluation of each water matrix wouldbe a challenge as many of the water samples are from dis-tant locations and duplicate samples were not possible. Tomonitor compound recovery and matrix interference,

Table 3Experimental mass balance correction factors for GC–MS/MScompounds

Compound Correction factor

GC–MS/MS

Acenaphthylene 0.81Aldrin 0.62Alpha-BHC 0.95Alpha-chlordane 0.69Anthracene 0.56Benz[a]anthracene 0.50Benzo[a]pyrene 0.50Benzo[b]fluoranthene 0.50Benzo[k]fluoranthene 0.50Beta-BHC 0.97Chrysene 0.50DDD 0.57DDE 0.62DDT 0.67Delta-BHC 0.96Dieldrin 0.83Endrin 0.88Fluoranthene 0.55Fluorene 0.67Galaxolide 0.75Gamma-BHC 0.97Gamma-chlordane 0.62Heptachlor 0.68Heptachlor epoxide 0.88Methoxychlor 0.70Metolachlor 0.94Mirex 0.66Musk ketone 0.87Naphthalene 0.91Phenanthrene 0.56Pyrene 0.55

Table 4DI and surface water with wastewater influence matrix spikes for GC–MS/MS compounds

Compound DI waterrecovery

Surface waterrecovery

Percentloss

GC–MS/MS % % %Acenaphthylene 69 72 0Aldrin 48 29 40Alpha-BHC 59 70 0Alpha-chlordane 64 47 27Anthracene 83 72 13Benz[a]anthracene 82 64 22Benzo[a]pyrene 67 52 22Benzo[b]fluoranthene 71 60 15Benzo[k]fluoranthene 69 59 14Beta-BHC 81 86 0Chrysene 77 65 16DDD 64 49 23DDE 65 32 51DDT 71 42 41Delta-BHC 74 78 0Dieldrin 66 68 0Endrin 82 84 0Fluoranthene 91 80 12Fluorene 71 67 6Galaxolide 30 30 0Gamma-BHC 65 78 0Gamma-chlordane 55 39 29Heptachlor 55 47 15Heptachlor Epoxide 74 71 4Methoxychlor 75 69 8Metolachlor 75 79 0Mirex 46 24 48Musk ketone 72 65 10Naphthalene 56 48 14Phenanthrene 84 74 12Pyrene 92 79 14

R.A. Trenholm et al. / Chemosphere 65 (2006) 1990–1998 1995

labeled surrogates are added prior to solid-phase extrac-tion. Surrogate recoveries outside of ±20% the mean werere-analyzed to ensure that the deviation was not instrumen-tal. Surrogate standards were used for the purpose of deter-mining possible analyte loss during sample extraction andprocessing. Surrogate recovery data was reported with eachtesting event. Although not every compound could bemonitored with an isotopically labeled standard, a varietyof compound classes were represented.

3. Results and discussion

Spike recovery experiments displayed a good degree ofreproducibility, with percent relative standard deviations(% RSD) of less than 22 for all compounds, and less than15 for the majority of compounds (Table 2). While recov-ery data were consistent, more volatile compounds hadlower recovery and greater variability. One example is gal-axolide, which had an average recovery of 30%. The lowrecovery was acceptable since environmental levels of thiscompound tend to be relatively high. In order to achieveideal recoveries for all compounds, more compound spe-cific methods would be required. However, the goal of thismethod was to develop a single method that encompassed a

wide range of compounds for rapid screening of variousaqueous matrices and removal through different treatmentprocesses. Although at pH 2 the basic compounds wouldbe charged, they still possessed enough hydrophobic inter-actions with the HLB to be retained and subsequently berecovered in the extracts. A few smaller basic compounds,such as cotinine, did not recover and were removed fromfurther evaluation.

Initial method development used a single 10 ml metha-nol elution. However, to increase steroid recoveries, a lesspolar solvent (MTBE) was added. Many of GC–MS/MScompounds also had good recoveries with methanol andMTBE elution. The PAHs, however, were very low tonone. A second elution (F2) was added using 10 ml ofDCM to improve recoveries of the PAHs. Liquid/liquidextraction was necessary to exchange the non-polar com-pounds from the methanol fraction and into a more suit-able solvent for GC–MS/MS analysis with the additionalbenefit of sample clean up.

During method development, it was discovered that cer-tain compounds (androstenedione, testosterone, progester-one, trimethoprim, acetaminophen, and fluoxetine) woulddegrade over time when spiked into unpreserved natural

1996 R.A. Trenholm et al. / Chemosphere 65 (2006) 1990–1998

water samples (Vanderford et al., 2003). Sulfuric acid andformaldehyde preservation were tested and comparedagainst a deionized water control. Sulfuric acid adequatelypreserved the samples without affecting compound recover-ies, while formaldehyde preservation still resulted in theloss of certain compounds (e.g. dilantin, iopromide, TCEP,and acetaminophen).

For the analysis of treated drinking water samples, oxi-dant (chlorine, chloramine, and ozone) residuals requiredquenching to stop further reactions during storage. Threecommon quenching agents were compared: sodium thiosul-fate (Na2S2O3), ammonium chloride (NH4Cl), and ascorbicacid. Tests were initially performed using spiked deionizedwater samples to determine the effects of each quenchingagent on compound recoveries. Sodium thiosulfate resultedin significant loss of trimethoprim, erythromycin, fluoxe-tine, atrazine, diazepam, progesterone and diclofenac.Ascorbic acid resulted in a 50% loss of only erythromycin,

Table 5Occurrence of target compounds in influent and treated wastewater (ng l�1)

Compound Nevada tertiaryeffluent

Korea membraneeffluent

Korefflu

Beta-BHC 11 nd ndFluorene nd 15 ndGalaxolide 395 715 720Gamma-BHC nd 15 10Metolachlor nd nd 11Mirex nd nd ndMusk ketone 37 217 212Naphthalene nd nd ndPhenanthrene nd nd ndAndrostenedione 1.5 nd 3.5Estradiol nd nd ndEstriol 7.8 nd 15Estrone 20 nd 7.0Ethynylestradiol nd nd ndTestosterone 1.1 nd ndAcetaminophen 1.6 nd 1.8Atrazine 1.0 nd ndCaffeine 29 nd 111Carbamazepine 129 nd 145DEET 383 nd 16Diazepam 2.3 nd ndDiclofenac 3.3 nd 45Dilantin 106 nd 16Erythromycin 51 32 136Fluoxetine nd nd 1.7Gemfibrozil 5.7 nd 3.9Hydrocodone 68 nd 13Ibuprofen 5.6 nd 41Iopromide 4.6 152 2670Meprobamate 424 nd ndNaproxen 5.1 nd 53Oxybenzone nd 72 16Pentoxifylline nd nd 4.2Sulfamethoxazole 187 160 189TCEP 354 297 346Triclosan 4.1 50 17Trimethoprim 6.6 nd 71

nd = non-detect.

while ammonium chloride did not affect compound recov-eries. Even with the loss of erythromycin, ascorbic acid waschosen as the preferred quenching agent as it was mostcompatible with the sulfuric acid preservation. Selectedcompound recoveries for each quenching agent are shownin Supplementary material 6.

This research method was developed and used to evalu-ate the removal of various organic contaminants throughwastewater and drinking water treatment processes. Initialoccurrence tests were important to ensure that the chosentarget compounds were detected in the environment at rel-evant levels. Natural and treated water samples were ana-lyzed from a variety of sources, including raw andfinished drinking water, surface and ground water,and raw and treated wastewater. Results for several rawand treated wastewater samples from South Korea andthe US are shown in Table 5. Compounds that were notdetected above the reporting limits are not shown. In raw

ea secondaryent

California 1 rawinfluent

California 2 rawinfluent

nd nd13 nd797 40810 ndnd nd27 nd23 10426 nd17 ndnd 52nd 33252 137nd 49nd ndnd 4723500 14200nd nd64500 32500274 78700 154nd ndnd nd170 51628 79nd 10nd 1220nd 3568700 2270nd 17560 12417200 4480924 657nd nd320 360535 2443780 564716 213

R.A. Trenholm et al. / Chemosphere 65 (2006) 1990–1998 1997

wastewater, only 100 ml of water was extracted, since tar-get compound levels were expected to be much greaterand to minimize possible suppression of LC–MS/MS ana-lyses (Vanderford et al., 2003).

Supplementary material 4 lists compounds that weredetected in finished drinking water samples from Nevadaand Georgia, USA. Compounds detected include atrazine,caffeine, DEET, dilantin, gemfibrozil, ibuprofen, iopro-mide, meprobamate, and TCEP with the highest reportedconcentration at 29 ng l�1 for atrazine in drinking water.These results demonstrate that several compounds weredetectable and suggest that some compounds have the abil-ity to penetrate through drinking water treatment pro-cesses.

Various surface and ground waters matrices were alsotested using this method. The amount of samples testedand their data is beyond the scope of this paper; however,a few examples are shown, including aquifer storage waterfrom Australia and river water from Kentucky (Ohio River)and Nevada (Colorado River). Only detected compoundsare listed in Supplementary Material 5 and include galaxo-lide, musk ketone, andronestenedione, estriol, ethynylestra-diol, atrazine, caffeine, carbamazepine, DEET, diazepam,dilantin, erythromycin, gemfibrozil, ibuprofen, iopromide,meprobamate, naproxen, sulfamethoxazole and TCEP.Reported values ranged from 1.1 to 571 ng l�1, with thehighest reported value for atrazine in the Ohio River water.

4. Conclusions

With the growing attention to EDCs and pharmaceuti-cals in the environment there is need for quick and sensi-tive analytical methods that can screen for a wide varietyof compounds simultaneously. This method allows for thetesting of several pharmaceuticals, pesticides, PAHs, ste-roids, and personal care products with one extractionand limited sample preparation. Using LC–MS/MS andGC–MS/MS methodologies, a wide range of organic con-taminants can be analyzed in many types of aqueousmatrices with the sensitivity and selectivity necessary foridentification and quantification at environmentally rele-vant levels.

Acknowledgements

This work was supported in part by the American WaterWorks Association Research Foundation (AwwaRF)project# 2758 entitled ‘‘Evaluation of Conventional andAdvanced Treatment Processes to Remove Endocrine Dis-ruptors and Pharmaceutically Active Compounds’’. Theauthors wish to acknowledge Dr. Paul Westerhoff of Ari-zona State University and Dr. Yeomin Yoon of CH2MHill, for their grateful insight and contributions towardsthis investigation. The authors would also like to thankthe staff at the Southern Nevada Water Authority for theirassistance throughout this endeavor.

Appendix A. Supplementary data

Supplementary data associated with this article canbe found, in the online version, at doi:10.1016/j.chemosphere.2006.07.004.

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