Detection of the Antimicrobial Triclosan in Environmental Samplesby ImmunoassayKi Chang Ahn,†,§ Anupama Ranganathan,† Candace S. Bever,† Sung Hee Hwang,† Erika B. Holland,‡

Kevin Morisseau,† Isaac N. Pessah,‡ Bruce D. Hammock,† and Shirley J. Gee*,†

†Department of Entomology and Nematology and UCD Comprehensive Cancer Center and ‡Department of Molecular Biosciences,University of California Davis, Davis, California 95616, United States

*S Supporting Information

ABSTRACT: A sensitive, competitive enzyme-linked immu-nosorbent assay (ELISA) for the detection of the antimicrobialtriclosan (TCS; 2,4,4′-trichloro-2′-hydroxydiphenyl ether) wasdeveloped. Novel immunizing haptens were synthesized byderivatizing at the 4-Cl position of the TCS molecule.Compounds derived from substitutions at 4′-Cl and thatreplaced the 2′-OH with a Cl atom were designed as uniquecoating antigen haptens. Polyclonal rabbit antisera werescreened against the coating antigen library to identifycombinations of immunoreagents resulting in the mostsensitive assays. The most sensitive assay identified was oneutilizing antiserum no. 1155 and a heterologous competitivehapten, where the 2′-OH group was substituted with a Clatom. An IC50 value and the detection range for TCS in assay buffer were 1.19 and 0.21−6.71 μg/L, respectively. The assay wasselective for TCS, providing low cross-reactivity (<5%) to the major metabolites of TCS and to brominated diphenyl ether-47. Asecond assay utilizing a competitive hapten containing Br instead of Cl substitutions was broadly selective for both brominatedand chlorinated diphenylethers. Using the most sensitive assay combination, we measured TCS concentrations in water samplesfollowing dilution. Biosolid samples were analyzed following the dilution of a simple solvent extract. The immunoassay resultswere similar to those determined by LC−MS/MS. This immunoassay can be used as a rapid and convenient tool to screen forhuman and environmental exposure.

■ INTRODUCTION

Triclosan (TCS; 2,4,4′-trichloro-2′-hydroxydiphenyl ether) iswidely used as an antimicrobial agent in household andpersonal care products (Figure 1). The widespread use of TCShas resulted in its presence in wastewater effluents, biosolids,and in surface receiving waters.1−5 This results in directexposure to aquatic animals, such as fish and snails. The landapplication of biosolids presents concerns about the potentialfor reentry of TCS into the environment and, therefore,additional exposure pathways.6 Human and animal exposure toTCS is of great concern because it has been demonstrated to bean antagonist in both estrogen-mediated and androgen-mediated bioassays and a potent Ca2+ channel sensitizer anduncoupler in a ryanodine receptor-mediated bioassay andprimary muscle cells, and it increases susceptibility to livercarcinogenesis.7−10

Current analytical methods are based on LC−MS/MS, GC/ECD, or GC/MS for the detection of TCS in wastewater andenvironmental samples. Analysis of water and biosolids typicallyincludes sample preparation steps such as liquid−liquid andsolid-phase extraction and, for GC analysis, derivatization of thehydroxyl group.11−13 Although these methods are well-suitedfor their applications, for routine monitoring, a simple, robust,

rapid method that can analyze a large number of samples isdesired. Immunoassay methods can serve as a rapid screen forenvironmental contaminants, pesticides, and their degradationproducts in environmental chemistry.14,15 These techniques arewidely used in diagnostics, environmental monitoring, foodquality, agriculture, and field or on-site testing of personnelexposed to toxic chemicals. We have demonstrated theseroutine immunoanalytical techniques using environmental andbiological samples such as house dust, soil, water, urine, andblood.14,16,17

The objective of this study was to develop an enzyme-linkedimmunosorbent assay (ELISA) for the analysis of TCS as asimple monitoring tool. Our approach was 2-fold. First, novelimmunizing and competitive haptens were carefully designedand synthesized. For immunizing, the design focused onhaptens that most closely mimicked the target analyte for thedevelopment of selective antibodies. With competitive haptens,we designed haptens that should have relatively lower affinity

Received: November 12, 2015Revised: February 17, 2016Accepted: March 2, 2016Published: March 3, 2016

Article

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compared to the target analyte to improve the sensitivity of theassay. Second, we took advantage of the strengths of using thehapten−protein-coated format. Advantages of this formatinclude using rabbit antibody sparingly compared to theantibody-coated format that generally used 10 to 100 timesmore antibody reagent. Hapten, which can be costly tosynthesize, is also used more sparingly in this format becausehapten−protein conjugates as coating antigens are generallymore stable than hapten−enzyme conjugates that require thepreservation of the enzymatic activity to be functional.Immunoassays for TCS detection have been developed

previously.18,19 However, the formats in which these have beendeveloped require a relatively large amount of primary antibodyreagent because the solid support (e.g., magnetic beads ormicrotiter plate) is coated with primary antibody. Becausepolyclonal antibody reagents are typically only generated oncefrom a single animal, their supply is limited, thus not permittingits use for analyzing large sample sets without the potentialneed to reoptimize another aliquot of reagent. An ELISAformat in which a labeled secondary antibody is used wouldpermit a more sparing use of primary antibody while using ahapten−protein conjugate as the immobilized coating antigen,thereby extending the usefulness of the assay.This study reports the preparation of novel haptens,

characterization of newly generated antibodies, immunoassayoptimization, and validation. This is the first report on theanalysis of biosolids by immunoassay. Furthermore, thereagents generated may be further applied to immunosensor-based assays and used in the generation of antibodies from newsources, such as camelid-derived VHH nanobodies, which areongoing projects in this laboratory.

■ EXPERIMENTAL SECTIONChemicals and Instruments. All hapten coupling reagents

(sulfo-N-hydroxysuccinimide, N-hydroxysuccinimide, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide, dicyclohexylcarbodii-mide, isobutyl chloroformate, tri-n-butylamine), bovine serumalbumin (BSA), thyroglobulin (Tg), goat antirabbit IgGperoxidase conjugate (GAR-HRP), Tween 20, and 3,3′,5,5′-

tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). ELISA was performedon 96-well microtiter plates (Nunc MaxiSorp, Roskilde,Denmark) and read spectrophotometrically with a microplatereader (Molecular Devices, Sunnyvale, CA) in dual-wavelengthmode (450 and 650 nm).

Hapten Synthesis. Due to their molecular size, TCShaptens require conjugation to carrier proteins to beimmunogenic. Thus, TCS haptens containing a carboxylicgroup or an amino group were designed and synthesized(Figure 1). The −NH2 or −COOH linker was introduced atthe 4-Cl (hapten T2 or T3) position in the TCS molecule tokeep the −OH group free and most distal to the point ofattachment to the protein. To retain the −OH group, weprotected it during synthesis of the haptens and thendeprotected it using boron tribromide. When coupled to thecarrier protein, these haptens fully presented the majority of theTCS molecule for antibody recognition resulting in an antibodyselective for TCS.Hapten precursors described in the Supporting Information

were initially synthesized with a nitroaromatic (4; Scheme 2) oraldehyde (7, Scheme 3 ) analog prepared from commerciallyavailable starting materials via nucleophilic aromatic substitu-tion. Compound 4 was hydrogenated to provide the aniline (5,Scheme 2 ), followed by boron tribromide-mediated cleavage ofthe methyl ester to obtain the immunizing hapten T2 that wasconjugated to protein by the diazotization method.An unsaturated spacer was introduced into methylated

dichlorophenoxybenzaldehyde (7) by enylation with phospho-noacetate or phosphonocrotonate using lithium hydroxide andmolecular sieve by the Wittig or Horner−Wadsworth−Emmons reaction20 to synthesize the intermediates 8, 18, or20 (Schemes 3, 8, or 9, respectively). To produce hapten T3,we converted the unsaturated spacer in intermediate 8 to asaturated spacer (9) by using Pd/C under H2 atmosphere.Cleavage of the aryl ether (Ar−OCH3) and the ester by borontribromide provided a strategy for the unmasking of both theArOH and ArCO2H functional groups.21 For coating haptenT7, compound 8 was not converted to a saturated spacer. A

Figure 1. Structures of immunizing and coating haptens. Arrows on the TCS structure indicate sites where the functional group was introduced.Haptens B1 and B2 were previously used for the development of a BDE-47 immunoassay.14

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similar strategy was used for coating haptens T8 and T9(Figure 1) using compounds 18 and 20 with unsaturatedlinkers. All four haptens were coupled to proteins using the N-hydroxysuccinimide method.22

The haptens T1 and T5 described in Schemes 1 and 5 werealkylated to obtain different lengths of a single-bondedhydrocarbon linker at the 2′-OH position of the TCS molecule.Ethyl bromobutyrate or ethyl bromohexanoate was used for thecarbon linker attachment on the TCS structure. More detailedsynthetic procedures are provided in the SupportingInformation.Preparation of Immunogen and Coating Antigens.

For haptens containing a −COOH group, conjugation toproteins was made using the N-hydroxysuccinimide (NHS),22

sulfo-NHS carbodiimide,22 or mixed anhydride method.23

Haptens containing an −NH2 group were conjugated by adiazotization method.22 Haptens T1, T2, and T3 (Figure 1)conjugated to Tg were used as immunogens. Haptens T1−T9were conjugated to bovine serum albumin (BSA) for use ascoating antigens. Further hapten−protein conjugation detailscan be found in the Supporting Information.Immunization and Antiserum Preparation. The immu-

nization was performed according to the procedure reportedpreviously.24 For each immunogen, three female New Zealandwhite rabbits were immunized (rabbits 1288, 1289, and 1290against hapten T1−Tg, rabbits 1154, 1155, and 1156 againsthapten T2−Tg, and rabbits 1157, 1158, and 1159 againsthapten T3−Tg). After seven boosts, a final serum was collectedabout 5 months following the first immunization. Antiserumwas obtained by centrifugation, stored at −20 °C, and usedwithout purification.Immunoassay. The ELISA was performed according to the

procedure described previously.24 In this format, the hapten−protein conjugate is coated to the well of the microtiter plate.After incubation with analyte and primary antibody, unboundprimary antibody is washed away. The remaining primaryantibody is detected using a secondary antibody that isconjugated to an enzyme. The IC50 value, an expression ofthe sensitivity of the immunoassay, and the limit of detection(LOD), defined as the IC10 value, were obtained from a four-parameter logistic equation. Borosilicate glass tubes were usedto prepare standard and sample solutions.Cross-reactivity. The cross-reactivity studies were eval-

uated using compounds that are structurally similar to TCS.The cross-reactivity was calculated as the (IC50 of the targetanalyte/IC50 of the tested compound) × 100.Preparation of Water Samples. Water samples used for

TCS analysis were taken from a fish exposure studyrepresentative of U.S. Environmental Protection Agency(USEPA) standard exposure procedures.25 Water samplesused in the fish exposure study were prepared using a singlebatch of deionized water adjusted to USEPA moderately hardstandards (pH 7.4−7.8; hardness 80−100 mg/L; alkalinity 57−64 mg/L).26 Aliquots of the adjusted water were added tobeakers and then spiked with 100 μL of concentrated stocksolutions of TCS in methanol to obtain TCS concentrations of0−300 μg/L with a final concentration of 0.01% methanol.Aliquots from each beaker were taken for analysis prior to theaddition of fish. For analysis, water samples were diluted 5−25times with 10% methanol in phosphate-buffered saline (PBS)buffer to bring the absorbance values within the linear range forimmunoassay. For LC−MS/MS analysis, water samples were

extracted in triplicate by solid-phase extraction (SPE) asdetailed in the Supporting Information.

Analysis of Biosolid Samples. Biosolid samples werecollected from a regional wastewater-treatment plant thatprocesses about 140 million gallons of wastewater daily. At thetime of sample collection, this facility diverted about 20% of thetotal sewage sludge to the production of biosolids for use asfertilizer, while the remainder was disposed into unlineddedicated land disposal areas. Samples were obtained from thededicated land disposal area and were not further characterized.The samples were prepared according to the method ofOgunyoku and Young.27 Briefly, samples were dried at 70 °Cfor 24 h and homogenized with a mortar and pestle. Foranalysis by immunoassay, 15 mL of a mixture of methanol andacetone (1:1, v/v) were added to 1 g of dried sample. Themixture was shaken for 24 h at 210 rpm at 60 °C andcentrifuged for 30 min at 4000 rpm. The extract was furtherdiluted 375−3000 times with 10% methanol in PBS buffer priorto the immunoassay. For LC−-MS/MS analysis, the driedbiosolid samples (1.0 g) were extracted with methanol using areflux apparatus. The extract was purified by SPE prior to usingthe LC−MS/MS analysis method of Ogunyoku and Young.27

(see the Supporting Information for details).

■ RESULTS AND DISCUSSIONHapten Synthesis. Novel immunizing haptens that mimic

the whole TCS molecule were designed and synthesized(Figure 1). Other researchers have designed haptens that linkedthe TCS molecule to carrier proteins through the 2′-OHgroup.28 Our design focused on utilizing linkers that allowedthe 2′-OH group to remain free, as it is in the parentcompound, because the 2′-OH is one element thatdistinguishes TCS from other diphenyl ethers. Another studyexplored using haptens that mimic only part of the TCSmolecule but found that titers were low to these fragmentaryhaptens.19 Although we and others have utilized thisfragmentary strategy successfully for polybrominated diphenylethers and polychlorinated biphenyls,14,29 for this study wefocused on haptens that mimic the whole molecule forimmunization.In a competitive ELISA, reducing the apparent affinity of the

antibody for the coating antigen relative to its affinity for TCSusually results in more sensitive assays. Our coating haptendesign utilized several strategies for achieving this goal. Forexample, haptens T7, T8, and T9 contain linkers that areunsaturated, and immunizing haptens contained linkers thatwere saturated. All coating haptens contained substitutions atthe 2′-OH position, while immunizing haptens retained the 2′-OH. Linkers were attached at the 2′- or 4′- positions in coatinghaptens but were in the 4- position for immunizing haptens.Finally, haptens where the 2′-OH was substituted with Cl or Brwere also synthesized. Such changes in linker composition,linker location, and substitutions at key positions provide alibrary of coating antigens that can greatly improve the chancesof developing a highly sensitive and selective immuno-assay.30−32 The structural characterization of all haptens isprovided in the Supporting Information.

Antibody Characterization. The titer of the antiseracollected after each boost was determined by the homologousindirect ELISA. All of the antisera showed relatively highconstant titers after the fifth immunization and no significantaffinity for BSA alone. All nine antisera were then screened forinhibition against all coating antigens at two concentrations (5

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and 500 μg/L) of TCS. Antisera/coating antigen combinationsthat showed over 50% inhibition at 500 μg/L and over 20%inhibition at 5 μg/L were rescreened at 10 concentrations ofTCS ranging from 0.003 to 5000 μg/L.All antisera−coating antigen combinations that were

homologous assays had IC50 values above 750 μg/L or, inthe case of antiserum no. 1156, exhibited low binding, asdemonstrated by the low Amax. Among the heterologousantisera−coating antigen assays, those utilizing antisera againsthapten T1 (no. 1288) or T3 (no. 1158) had higher IC50 values,while antiserum against hapten T2 (no. 1155 and no. 1156)had mixed results depending upon the coating antigen used(Table 1). The assay demonstrating the lowest IC50 usedantiserum no. 1155 that was raised against immunizing haptenT2 and coating antigen T8-BSA. Similar to the results fromBrun and colleagues,19 the best immunogen identified was onethat contained a nitrogen as the first atom on the linking arm.Haptens B1 and B2 that replace chlorine substituents of TCS

hapten T8 by bromine atoms were synthesized to develop aBDE immunoassay (Figure 1).14 These haptens were screenedto introduce additional heterology in the coating antigen. Asimilar strategy was used for an earlier TCS assay28 and for anassay for deltamethrin.33 For antiserum no. 1288 (raised againstT1-Tg) and antiserum no. 1158 (raised against T3-Tg), the useof B1-BSA resulted in improved sensitivity compared to thebest TCS-based coating antigen for each antibody. Forantiserum no. 1155, both B1- and B2-BSA resulted in sensitiveassays but were not superior to assays utilizing TCS haptens T8and T9. Among the combinations of B1-BSA−antiserum no.1288, B1-BSA−antiserum no. 1158, and B1-BSA−antiserumno. 1155, the latter was selected as one of the assays used infurther assay development because it had the lowest overallIC50 among the assays utilizing brominated coating antigens(Table 1).Unique coating haptens in which the hydroxyl group was

replaced by a Cl atom (haptens T6, T8, and T9) for theheterologous competition format resulted in remarkablyincreased assay sensitivity. Although the antibody (no. 1155)bound these coating haptens with moderate affinity, the affinityfor free TCS was greater, allowing the displacement of theantibody by low concentrations of TCS resulting in a sensitive

assay. Conversely, the assay that used hapten T7 that containeda −OCH3 group in place of the −OH was not sensitive,indicating that the affinity of the antibody for the −OCH3

group was greater than for the −OH. In conclusion, the coatingantigen, hapten T9-BSA (where the hydroxyl group wasreplaced by a Cl atom and a medium-length rigid carbon linkerwas included), along with antibody no. 1155, was selected forfurther assay development due to its high maximum signal, highsignal-to-noise ratio, steep slope, and low IC50 value.

Optimization. Utilizing immunizing haptens T1 and T2resulted in reasonable antibody affinity to the target analyte in acompetitive assay format and rabbit antiserum no. 1155generated against T2 provided the lowest IC50 (Table 1).Hapten T9-BSA and antiserum no. 1155 was found to be thebest combination and was used for these optimization studies.Methanol (MeOH) is generally used as a cosolvent in the assaybuffer (PBS) to ensure the solubility of lipophilic analytes. Asseen in Table 2, because lower amounts of MeOH providedbetter sensitivity, 10% MeOH was selected for the further assaydevelopment. The IC50 was not significantly affected by pHvalues ranging from 5.5 to 8.5 in the buffer, but the maximum

Table 1. Characteristics of the ELISA Using Various Combinations of Antiserum and Coating Antigen

format immunogen antiserum no. coating antigen A/D

Amaxa slope IC50 (μg/L) Amin

homologous T1-Thy 1288 T1-BSA 1.03 1.00 788 0.17 6heterologous T1-Thy 1288 B1-BSAb 0.78 0.94 49.1 0.06 14heterologous T3-Thy 1158 T2-BSA 1.11 1.45 1240 0.31 4heterologous T3-Thy 1158 B1-BSAb 1.26 1.58 488 0.04 33heterologous T3-Thy 1158 B2-BSAb 1.11 1.45 1240 0.31homologous T2-Thy 1155 T2-BSA 0.26 0.40 1950 −0.02 16heterologous T2-Thy 1155 T3-BSA 0.75 0.24 1590 −0.24 3heterologous T2-Thy 1155 B1-BSAb 0.72 0.71 15.4 0.23 3heterologous T2-Thy 1155 B2-BSAb 0.92 0.68 20.7 0.28 3heterologous T2-Thy 1155 T6-BSA 0.34 0.69 3.8 0.05 6heterologous T2-Thy 1155 T8-BSA 0.86 0.60 1.5 0.07 13heterologous T2-Thy 1155 T7-BSA 0.69 0.21 8010 −0.01 62heterologous T2-Thy 1155 T9-BSA 0.89 0.86 2.0 0.07 12homologous T2-Thy 1156 T2-BSA 0.16 0.98 8 0.05 3heterologous T2-Thy 1156 T3-BSA 0.36 1.06 24 0.06 6heterologous T2-Thy 1156 B1-BSAb 0.12 0.54 327 0.02 6

aParameters calculated from a four-parameter logistic fit of a calibration curve. bFrom Ahn et al.14

Table 2. Effect of pH or Solvent on the Assay Sensitivity

parameters derived from the four-parameter curve fita

Amax slope IC50 (μg/L) Amin A/D

solvent effect10% MeOH 0.896 1.07 63.0 0.132 720% MeOH 0.985 0.89 67.5 0.130 840% MeOH 0.997 0.94 120 0.135 7pH effectpH 5.5 1.10 1.12 2.1 0.088 13pH 7.4 1.11 1.02 1.6 0.103 11pH 8.5 0.940 0.81 1.2 0.059 16pH 10.7 0.789 0.83 1.6 0.076 10

aAmax is the absorbance observed in the absence of analyte, IC50 is thecalculated valued described as the concentration resulting in a 50%decrease in maximum signal, and Amin is the absorbance observed atmaximum inhibition of signal. Hapten B1-BSA and antiserum no. 1288were used for the analysis of methanol effect.

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Figure 2. Optimized conditions and standard curve for TCS immunoassay.

Table 3. Cross-Reactivity (%) of Compounds Structurally Related to TCS

aNI = no inhibition at 5000 μg/L.

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absorbance signals varied. A pH 8.5 PBS assay buffer containing10% MeOH was selected for further experiments because theassay had the lowest IC50 and a good ratio of maximum tominimum signal (A/D) of 16. Because the maximumabsorbance signals are generally significantly reduced by higherionic strength (2× or 4× PBS) due to the suppression of thebinding interaction of antibody to antigen, 1× PBS (0.15 M)was used for the assay.The final assay conditions were as follows: Primary antiserum

no. 1155 was diluted in PBST (PBS containing 0.05% Tween20). The optimized ELISA used a coating antigen of T9-BSA ata concentration of 1 μg/mL and antiserum no. 1155 producedagainst hapten T2-Tg at a dilution of 1/5000 in PBST beforethe competition. The plate coated with the coating antigen wasblocked with 0.5% BSA. The assay buffer was 10% MeOH in0.15 M PBS, pH 8.5. Under these conditions, the assay had alinear range (IC20−80) of 0.2−6.7 μg/L of the target and an IC50value of 1.2 μg/L. The LOD in buffer was 0.1 μg/L, the IC10value (Figure 2). This assay is comparable in sensitivity to theformats reported earlier that exhibited IC50 values for TCS of3.8519 and 0.25 μg/L18

Cross-reactivity. TCS metabolites and its structural analogswere evaluated for cross-reactivity. The assay was selective forthe target analyte TCS. Cross-reactivities to methyl TCS(2,4,4′-trichloro-2′-methoxydiphenyl ether) and BDE-47(2,2′,4,4′-tetrabromodiphenyl ether) were 3 and 4%, respec-tively, while the antimicrobial triclocarban and two thyroidhormones cross-reacted <1% (Table 3). These cross-reactivitypatterns are very similar to the assay described by Brun et al.,19

and the assay by Shelver18 cross-reacted with methyl-TCS(312%) and BDE congeners (2.5−64%). It is likely that boththe phenyl and hydroxyl are important for binding by theantibody that was generated against immunizing hapten T2,where the −OH was exposed, and explains the similar findingsby Brun et al. in which the phenyl and hydroxyl are alsoexposed.19 The antibody generated against hapten T1, wherethe −OH group of TCS was alkylated to −OCH2(CH2)2−COOH, highly recognized methyl TCS and BDE-47, andshowed less specificity to TCS due to the lack of H-bondingdonation, which is consistent with earlier findings using asimilar immunizing hapten conjugated through the hydroxyl.18

Analysis of Fish Exposure and Biosolid Samples.Watersamples that were used to test the toxicity of TCS to fish werediluted 5-fold at low concentrations and 25-fold at highconcentrations prior to the immunoassay to bring the samplesinto the linear range of the assay. Recoveries of 85−92% wereobtained from both the immunoassay and LC−MS/MSanalysis. The recoveries obtained using the immunoassaymethod are comparable to other studies in which fortifiedmineral and wastewater samples were analyzed withoutcleanup,18,19 having reported recoveries ranging from 75 to113%. Similarly, recoveries above 85% were reported for LC−MS/MS methods that utilized solid-phase extraction.34,35 Anaverage ratio between instrumental analysis and immunoassayresults was less than 1.2 (Table 4).Analysis of biosolid samples can be challenging because of

the complexity of the samples; thus, multistep sampleextraction and cleanup methods are necessary,36 usingtechniques such as accelerated solvent extraction coupled tosolid-phase cleanup.37 Immunoassays are often advantageousbecause the antibody provides some selectivity for the analytein the presence of matrix, eliminating the need for exhaustivecleanup before analysis.38 Our goal was to use a simple, field-

portable sample preparation method; thus, samples wereextracted with methanol−acetone and shaking. For instrumen-tal analysis, the samples were extracted using a methanol refluxmethod described in the Supporting Information. The LC−MS/MS extraction and analysis showed higher levels of TCSthan the ELISA method. Because the sample preparationmethods were not identical, it is not known whether thedifference is due to extraction efficiency or a matrix effect oneither or both detection methods. Nevertheless, the concen-trations of TCS found in these biosolid samples were within thewide range of TCS concentrations found in other biosolids.39

The average ratio between the results of both methods was lessthan 1.6 (Table 5), indicating that this immunoassay is suitablefor the determination of TCS in biosolid samples as an alarm orprimary screening method.

In conclusion, for the development of a sensitive andselective immunoassay for TCS, a hapten preserving the 2′-OHgroup in a near-perfect molecular mimic of TCS aids in theselective recognition of the TCS by the resulting antibody. Aunique feature of this immunoassay is the use of a novelheterologous coating hapten, where the −OH was substitutedwith a −Cl. This resulted in a decreased affinity of the antibodyfor the coating antigen and subsequently increased the ability ofthe analyte to compete resulting in the highest sensitivity assay.This immunoassay was more sensitive (IC50 value of 1.2 μg/L)compared to the TCS assay developed by Brun et al. (3.85 μg/L)19 but less sensitive than the existing Abraxis assay kit forTCS (0.25 μg/L, http://www.abraxiskits.com). Moreover, thisimmunoassay utilizes less primary antibody than the previouslydescribed assays.18,19 Furthermore, the results from this assaywere in agreement with the LC−MS/MS method for testing

Table 4. TCS Concentrations in Water Samples by ELISAand LC−MS/MS

TCS measured (μg/L)

TCS added(μg/L) immunoassay LC−MS/MS

ratio (LC−MS/MS/immunoassay)

0 0.38 ± 0.14 0.54 ± 0.08 1.423 2.36 ± 0.70 3.05 ± 0.91 1.2930 26.9 ± 3.2 25.6 ± 1.07 0.95100 73.6 ± 13.0 84.0 ± 1.31 1.14300 298 ± 20.4 287.6 ± 6.45 0.96average recovery(%)

85.3 ± 11.6 91.7 ± 8.47 1.15 ± 0.20

Samples containing spiked concentrations of 0−3 μg/L were dilutedfive times in the assay buffer prior to analysis; samples containing100−300 μg/L were diluted 25 times in the assay buffer.

Table 5. TCS Concentrations in Biosolid Samples by ELISAand LC−MS/MS

TCS concentrationdetermined in biosolids (ng/g

dry weight)

sample immunoassay LC−MS/MSratio (LC−MS/MS/

Immunoassay)

DLD 2 966 ± 68 1720 ± 247 1.78DLD 3 392 ± 39 521 ± 102 1.33DLD 4 489 ± 20 826 ± 223 1.69average 1.60 ± 0.24

Immunoassay data is the mean of triplicate extractions. LC−MS/MSdata is the mean of two replicates.

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environmental samples with fewer, simpler extractions stepsneeded and is the first report of the analysis of TCS in biosolidsby immunoassay.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.est.5b05357.

Additional details include schemes and experimentaldetails on hapten synthesis, immunization, couplingmethods and an instrumental analysis of water andbiosolid samples. (PDF)

■ AUTHOR INFORMATIONCorresponding Author*Phone: 530-752-8465; fax: 530-752-1537; e-mail: sjgee@ucdavis.edu.Present Address§PTRL West (a Division of EAG Inc.), 625-B Alfred NobelDrive, Hercules, CA 94547NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis research was supported in part by the National Institute ofEnvironmental Health Sciences Superfund Basic ResearchProgram, P42 ES04699, the UC Davis Environmental HealthSciences Core Center, P30 ES023513, and the NationalInstitute of Occupational Safety and Health Western Centerfor Agricultural Health and Safety, 2U54 OH007550.

■ ABBREVIATIONSAhR aryl hydrocarbon receptorBDE polybrominated diphenyl etherBSA bovine serum albuminECD electron-capture detectorELISA enzyme linked immunosorbent assayGAR-HRP goat antirabbit IgG peroxidase conjugateGC gas−liquid chromatographyHPLC high-performance liquid chromatographyLC−MS/MS liquid chromatography tandem mass spectrom-

etryLOD limit of detectionMeOH methanolMS mass spectrometryNHS N-hydroxysuccinimidePBS phosphate buffered salinePBST phosphate buffered saline containing 0.05%

Tween 20SPE solid-phase extractionTCS triclosanTg thyroglobulinTMB 3,3′5,5′-tetramethylbenzidine

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S-1

Supporting Information

Detection of the Antimicrobial Triclosan in Environmental Samples by Immunoassay

Ki Chang Ahn†§, Anupama Ranganathan†, Candace S. Bever†, Sung Hee Hwang†, Erika Holland‡, Kevin

Morisseau†, Issac N. Pessah‡, Bruce D. Hammock† and Shirley J. Gee†*

Department of Entomology and Nematology and UCD Comprehensive Cancer Center†; Department of

Molecular Biosciences‡, University of California Davis, Davis, CA 95616

§Current address: PTRL West (a Division of EAG Inc.), 625-B Alfred Nobel Drive, Hercules, CA 94547

*Corresponding author phone: 530-752-8465, fax: 530-752-1537, e-mail: sjgee@ucdavis.edu.

Number of pages: 10

Number of figures: 9

Number of tables: 0

S-2

Hapten Syntheses

Chemicals and Instruments. All reagents were analytical grade from Fisher Scientific (Pittsburgh, PA) or Sigma-Aldrich (St. Louis, MO). Precoated silica gel 60 F254 glass plates (0.25 mm, EMD Chemicals, Temecula, CA) were used for thin layer chromatography (TLC) analysis. Silica gel was used for column chromatography. Proton NMR (1H NMR) spectra were measured with a General Electric QE-300 spectrometer (Bruker NMR, Billerica, ME) using tetramethylsilane as an internal standard. Electrospray mass spectra of haptens in positive (MS-ESI+) or negative (MS-ESI-) mode were recorded by a Micromass Quattro Ultima triple quadrupole tandem mass spectrometer (Micromass, Manchester, UK). Rf values refer to TLC on the silica gel plates with visualization under exposure to either ultraviolet light (254 nm) or iodine vapor. Nomenclature. The nomenclature of haptens was designated with the aid of Chemdraw Ultra 11.0 (CambridgeSoft, Cambridge, MA). Immunizing haptens

Synthesis of 4-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)butanoic acid (Hapten T1, BDH 382-02, Scheme 1): The mixture of triclosan (400 mg, 1.38 mmol), 4-bromo-butyric acid tert-butyl ester (399 mg, 1.79 mmol), and potassium carbonate (286 mg, 2.07 mmol) in 2 mL of anhydrous DMF was reacted at 100 oC for 3 h. The resulting mixture was filtered to remove excess K2CO3 and HBr generated in the reaction. The filtrate diluted with 20 mL of ethyl acetate was washed twice with 20 mL of distilled water. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The residue was chromatographed on silica gel eluting with a mixture of ethyl acetate/hexane (1:2, v/v). Fractions containing pure product by TLC were stripped under high vacuum to obtain 179 mg of Compound 2 (BDH 382-01) as a transparent oil. TLC (ethyl acetate/hexane=1:10, v/v) Rf, 0.54. Trifluoroacetic acid (TFA) (0.5 mL) was added to the ester intermediate (BDH 382-01) and the mixture was allowed to stand at ambient temperature for 30 min. After the addition of 50 mL of distilled water and acidification with 6 N HCl to pH 2, the mixture was extracted twice with 50 mL of ethyl acetate. The combined organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The concentrate was recrystallized from a mixture of ethyl acetate and hexane to give Hapten T1 (391 mg, yield: 62%) as a white solid: mp 85-88 oC; TLC (ethyl acetate/hexane/acetic acid, 5:15:0.1, v/v/v) Rf, 0.28; MS-ESI- m/z

calcd for [M - H]- =C16H13Cl3O4, 373.99; observed, 373.05. 1H NMR (300 MHz, chloroform-d) δ 7.43 (dd, J = 2.4, 1.4 Hz, 1H), 7.11 – 7.09 (m, 1H), 7.08 – 7.07 (m, 1H), 6.96 (s, 1H), 6.95 (d, J = 3.0 Hz 1H), 6.64 (d, J = 8.8 Hz, 1H), 3.99 (t, J = 5.9 Hz, 2H), 2.32 (t, J = 7.2 Hz, 2H), 1.96 (p, J = 6.7 Hz, 2H).

Scheme 1

S-3

Synthesis of 2-(4-amino-2-chlorophenoxy)-5-chlorophenol (Hapten T2, BDH 382-42, Scheme 2): This hapten was synthesized according to the method described (Freundlich et al. 2005). To a solution of 4-chloro-2-methoxyphenol (1085 mg, 6.84 mmol), suspended K2CO3 (945 mg, 6.84 mmol) in dimethylformamide (DMF, 5 mL) was added 3-chloro-4-fluoronitrobenzene (1000 mg, 5.7 mmol). The reaction mixture was refluxed overnight. The mixture was diluted with ethyl acetate (200 mL) and 1 N NaOH (200 mL). The organic layer was separated, washed with water (100 mL), and concentrated. The residue was recrystallized from a mixture of ethyl acetate and hexane to give a white solid, Compound 4 (BDH-382-26) (1539 mg, yield 86%). 1H NMR (300 MHz, chloroform-d) δ 8.36 (d, J = 2.7 Hz, 1H), 8.00 (dd, J = 9.1, 2.7 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 7.03 (d, J = 2.1 Hz, 1H), 7.02 – 6.98 (m, 1H), 6.67 (d, J = 9.1 Hz, 1H), 3.78 (s, 3H). The mixture of Compound 4 (500 mg, 1.60 mmol) and stannous chloride dihydrate (3610 mg, 16 mmol) in 10 mL of ethanol was stirred at 70 °C in an oil bath for 1 h and at room temperature for 2 h. The mixture was cooled and poured into the slurry of water, ethyl acetate, and Celite. NaHCO3 was added in portions. The neutral mixture was filtered, and the solids on the filter and in the flask were washed with water and ethyl acetate. The ethyl acetate phase was separated and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. The oil residue was purified by using silica gel chromatography with a mixture of methanol/methylene chloride (1:15, v/v) as an eluent to give Compound 5 (BDH-382-36) (411 mg, yield: 91%) as a brown solid. TLC (methanol/methylene chloride, 1:15, v/v) Rf , 0.78. 1H NMR (300 MHz, chloroform-d) δ 6.94 (dd, J = 2.3, 1.2 Hz, 1H), 6.83 – 6.81 (m, 1H), 6.80 – 6.78 (m, 0H), 6.77 (dd, J = 2.7, 1.2 Hz, 1H), 6.59 – 6.54 (m, 1H), 6.52 (dd, J = 2.8, 1.2 Hz, 0H), 3.89 (d, J = 1.2 Hz, 4H), 3.65 (s, 3H).

A solution of Compound 5 (350 mg, 1.24 mmol) in 20 mL CH2Cl2 was treated dropwise with 25 mL BBr3 in CH2Cl2 (1 M). The mixture was stirred at 0oC for 30 min and at room temperature overnight. Water (200 mL) and CH2Cl2 (twice, 100 mL) was added into the mixture. The organic layer was separated, combined, evaporated and recrystallized from H2O to get a brown solid of Compound 6 (BDH-382-42) (206 mg, yield: 62%): TLC (methanol/methylene chloride, 1:15, v/v) Rf, 0.53; MS-ESI+ m/z calcd for [M + H]+ =C12H9Cl2NO2, 269.00; observed, 270.03. 1H NMR (300 MHz, chloroform-d) δ 6.73 (d, J = 2.3 Hz, 1H), 6.61 (d, J = 4.7 Hz, 1H), 6.49 (dd, J1 = 3 Hz, J2 = 9 Hz, 1H), 6.39 (dd, J1 = 3 Hz, J2 = 9 Hz, 1H), 6.33 (dd, J1 = 3 Hz, J2 = 6 Hz, 1H), 6.26 (dd, J1= 1.8 Hz, J2 = 8.4 Hz, 1H).

Scheme 2

S-4

Synthesis of 3-(3-chloro-4-(4-chloro-2-hydroxyphenoxy)phenyl)propanoic acid (Hapten T3, BDH 382-48,

Scheme 3):

3-Chloro-4-(4-chloro-2-methoxyphenoxy)benzaldehyde (Compound 7, BDH-382-44) was synthesized according to the method described (Ahn et al. 2009) using 3-chloro-4-fluorobenzdehyde (1000 mg, 6.3 mmol) and 4-chloro-2-methoxyphenol (999 mg, 6.3 mmol). 1H NMR (300 MHz, chloroform-d) δ 9.87 (s, 1H), 7.97 (s, 1H), 7.64 (dd, J = 8.5, 2.0 Hz, 1H), 7.03 – 7.01 (m, 2H), 7.00 (d, J = 2.3 Hz, 1H), 6.72 (d, J = 8.5 Hz, 1H), 3.78 (s, 3H). To a solution of Compound 7 (500 mg, 1.69 mmol) and triethyl 4-phosphonoacetate (417 mg, 1.86 mmol) in dry tetrahydrofuran (THF, 10 mL) was added LiOH.H2O (78 mg, 1.86 mmol) and molecular sieve 4 Å (2.5 g). The mixture was refluxed overnight under N2 conditions. Ethyl acetate (50 mL), water (50 mL) and NaCl (1 g) were added and the organic layer was separated. After removing the organic solvent, the crude residue was purified by silica gel chromatography using a mixture of ethyl acetate and hexane (1:10, v/v) to give Compound 8 (BDH-

382-45) as a white solid (575 mg, yield: 93%): TLC (ethyl acetate/hexane=1:5, v/v) Rf, 0.51; 1H NMR (300 MHz, chloroform-d) δ 7.66 – 7.52 (m, 1H), 6.99 (q, J = 1.4 Hz, 1H), 6.97 – 6.90 (m, 1H), 6.67 (dd, J = 8.5, 1.8 Hz, 1H), 6.37 – 6.30 (m, 1H), 4.26 (dd, J = 7.0, 1.8 Hz, 2H), 3.80 (d, J = 1.8 Hz, 3H), 1.33 (td, J = 7.2, 1.9 Hz, 3H).

Compound 8 (130 mg, 0.36 mmol) was converted to Compound 9 (BDH-382-46) using Pd/C in ethanol (7 mL) at 70 oC under H2 condition. Pd/C was removed by filtration. The mixture was concentrated and the crude residue was purified by silica gel chromatography using a mixture of ethyl acetate and hexane (1:5, v/v) to give Compound 9 (50 mg, yield: 38%) as a transparent oil. TLC (ethyl acetate/hexane, 1:5, v/v, developed twice) Rf, 0.50. 1H NMR (300 MHz, chloroform-d) δ 7.28 (d, J = 2.1 Hz, 1H), 7.13 (s, 1H), 7.03 – 6.91 (m, 1H), 6.89 (dd, J = 5.1, 1.9 Hz, 2H), 6.86 (d, J = 1.9 Hz, 1H), 6.73 (d, J = 8.4 Hz, 1H), 4.13 (dd, J = 7.1, 2.2 Hz, 2H), 3.85 (d, J = 2.6 Hz, 3H), 2.90 (td, J = 7.7, 3.7 Hz, 2H), 2.60 (td, J = 7.7, 1.4 Hz, 2H), 1.24 (td, J = 7.2, 1.7 Hz, 3H).

To a cooled solution of Compound 9 (50 mg, 0.13 mmol) in CH2Cl2 was added a 1 M solution of BBr3 in CH2Cl2 (0.26 mL, 0.26 mmol). After stirring at 0 oC for 4 h, an additional 2 mL BBr3 in CH2Cl2 was added. The reaction mixture was stirred for another 30 min and then water (50 mL) and 1 N NaOH (3 mL) were added. The mixture was diluted with ethyl acetate (twice, 50 mL) and water (50 mL). The organic layers were separated, combined, and concentrated. The crude residue was recrystallized from a mixture of ethyl acetate and hexane to give a white solid of Compound 10 (BDH 382-48) (43 mg, yield 90%): TLC (methanol/methylene chloride/acetic acid, 2:18:0.02, v/v/v) Rf, 0.32; MS-ESI- m/z calcd for [M - H]- =C15H12Cl2O4, 326.01; observed, 325.05. 1H NMR (300 MHz, Chloroform-d) δ 8.92 (d, J = 44.9 Hz, 1H), 7.66 (s, 1H), 7.04 (d, J = 7.8 Hz, 1H), 6.89 (s, 2H), 6.75 (d, J = 8.2 Hz, 2H), 6.08 (s, 1H), 2.78 (s, 1H).

Scheme 3

S-5

Coating haptens

Synthesis of 2-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)acetic acid (Hapten T4, BDH 382-16, Scheme 4): Compound 11 (BDH 382-15) was synthesized using tert-butyl bromoacetate using the same synthetic method as Compound 3. That is, the mixture of triclosan (400 mg, 1.38 mmol), tert-butyl bromoacetate (349 mg, 1.79 mmol), and potassium carbonate (286 mg, 2.07 mmol) in 2 mL of anhydrous DMF was reacted at 100 oC for 3 h. The resulting mixture was filtered to remove excess K2CO3 and HBr produced in the reaction. The filtrate, diluted with 20 mL of ethyl acetate was washed twice with 20 mL of distilled water. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by evaporation. The residue was chromatographed on silica gel eluting with the mixture of ethyl acetate/hexane (1:2, v/v). Fractions containing pure product by TLC were stripped under high vacuum to obtain Compound 11 as a transparent oil. TLC (ethyl acetate/hexane=1:10, v/v) Rf, 0.47. Trifluoroacetic acid (TFA) (0.5 mL) was added to the ester intermediate (Compound 11) and the mixture was allowed to stand at ambient temperature for 30 min. After the addition of 50 mL of distilled water and acidification with 6 N HCl to pH 2, the mixture was extracted twice with 50 mL of ethyl acetate. The combined organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by evaporation. The concentrate was recrystallized from a mixture of ethyl acetate and hexane to give Hapten T4 (BDH 382-16) (436 mg, yield: 90%) as a white solid. mp 85-88 oC. TLC (ethyl acetate/hexane/acetic acid, 5:15:0.1, v/v/v) Rf, 0.20. 1H NMR (300 MHz, chloroform-d) δ 7.45 (d, J = 2.5 Hz, 1H), 7.14 (dd, J = 8.8, 2.5 Hz, 1H), 7.01 (d, J = 2.3 Hz, 1H), 7.00 – 6.98 (m, 1H), 6.89 – 6.82 (m, 1H), 6.79 (d, J = 8.8 Hz, 1H), 4.71 (s, 2H).

Scheme 4

Synthesis of 6-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)hexanoic acid (Hapten T5, BDH 382-17, Scheme 5): The mixture of triclosan (824 mg, 2.76 mmol), ethyl bromohexanoate (800 mg, 3.58 mmol), and potassium carbonate (572 mg, 4.14 mmol) in 2 mL of anhydrous DMF was reacted at 100 oC overnight. The resulting mixture was filtered to remove excess K2CO3 and HBr produced in the reaction. The filtrate diluted with 20 mL of ethyl acetate was washed twice with 20 mL of distilled water. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by evaporation. The residue was chromatographed on silica gel eluting with the mixture of ethyl acetate/hexane (1:2, v/v). Fractions containing pure product by TLC were stripped under high vacuum to the bromoester as a transparent oil. TLC (ethyl acetate/hexane, 1:10, v/v) Rf, 0.75. 6N NaOH (5 mL) and methanol (10 mL) was added to the ester intermediate and the mixture was allowed to stand at 70 oC for 2 d. After the addition of 50 mL of distilled water and acidification with 6 N HCl to pH 2, the mixture was extracted twice with 50 mL of ethyl acetate. The combined organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by evaporation. The concentrate was recrystallized from a mixture of ethyl acetate and hexane to give Hapten T5 (1022 mg, yield: 92%) as a white solid. TLC (methylene

S-6

chloride/MeOH/acetic acid=2:18:0.02, v/v/v) Rf, 0.54. 1H NMR (300 MHz, chloroform-d) δ 7.42 (d, J = 2.5 Hz, 1H), 7.09 (dd, J = 2.5, 0.9 Hz, 1H), 7.06 (dd, J = 2.5, 0.9 Hz, 1H), 6.96 – 6.95 (m, 1H), 6.94 (s, 4H), 6.93 (d, J = 1.0 Hz, 1H), 3.91 (t, J = 6.1 Hz, 4H), 2.29 (t, J = 7.4 Hz, 3H), 1.61 (dp, J = 22.8, 7.7, 7.1 Hz, 10H), 1.28 (q, J = 7.0, 5.6 Hz, 4H).

Cl

Cl

O

OH

Cl Cl

Cl

O

O

ClK2CO3, DMF

TCS

Hapten T5as coating hapten

O

O

Cl

Cl

O

O

Cl

O

OH

NaOH, H+

Br(CH2)5COOCH3

Scheme 5

Synthesis of 3-chloro-4-(2,4-dichlorophenoxy)benzoic acid (Hapten T6, BDH 382-63, Scheme 6): 2,4-Dichlorophenol (616 mg, 3.78 mmol) and 3-chloro-4-fluorobenzaldehyde (500 mg, 3.15 mmol) were dissolved in N,N-dimethylacetamide (DMAC, 10 mL). Anhydrous Na2CO3 and molecular sieve 4 Å were added and the reaction mixture was refluxed overnight under a stream of nitrogen gas. The mixture was diluted with ethyl acetate (200 mL) and 1 N NaOH (200 mL). The organic layer was separated, washed with water (100 mL), and concentrated. The crude residues were purified by silica gel chromatography using a mixture of ethyl acetate and hexane (1:20, v/v) to give Compound 15 (BDH-382-56) as a transparent oil (770 mg, yield: 82%). TLC (ethyl acetate/hexane, 1:5, v/v) Rf, 0.51. Compound 15 (770 mg, 2.57 mmol) was dissolved in dimethyl sulfoxide (5 mL) and tetrahydrofuran (5 mL). Silver oxide (1200 mg, 5.14 mmol) and water (5 mL) were added and the mixture was reacted at 100 oC for 2 h. The mixture was made alkaline with 1 N NaOH (50 mL) and washed with ethyl acetate to remove unreacted Compound 15. The alkaline solution was acidified with 6 N HCl and extracted with ethyl acetate. The organic layer was separated and concentrated. The residue was recrystallized from ethyl acetate and hexane to give Compound 16 (BDH-382-63) (522 mg, yield: 64%): TLC (methanol/methylene chloride/acetic acid, 2:18:0.02, v/v/v) Rf, 0.46; MS-ESI- m/z calcd for [M - H]- =C13H7Cl3O3, 315.95; observed, 314.9; 1H NMR (300 MHz, DMSO-d6) δ 13.25 (s, 1H), 8.04 (d, J = 2.1 Hz, 1H), 7.84 (dd, J = 8.4, 2.7 Hz, 1H), 7.50 (dd, J = 8.7, 2.4 Hz, 1H), 7.27 (d, J = 9 Hz, 1H), 6.93 (d, J = 8.7 Hz, 1H).

Scheme 6

Synthesis of (E)-3-(4-(2,4-dichlorophenoxy)-3-methoxyphenyl)acrylic acid (Hapten T7, BDH-382-68, Scheme

7)

Compound 8 was dissolved in MeOH and hydrolyzed under 10% alkaline conditions at 70 oC overnight. The reaction mixture was acidified with 6 N HCl and extracted with ethyl acetate. The organic layer was concentrated. The residue was recrystallized from a mixture of ethyl acetate and hexane to give Hapten T7 as a white solid (20

S-7

mg, yield: 8%). TLC (methanol/methylene chloride/acetic acid, 2:18:0.02, v/v/v) Rf, 0.43. 1H NMR (300 MHz, DMSO-d6) δ 12.40 (s, 1H), 7.93 (d, J = 1.5 Hz, 1H), 7.57 (d, J = 2.1 Hz, 1H), 7.51 (d, J = 15.9 Hz, 1H), 7.29 (d, J

= 2.1 Hz, 1H), 7.10 (d, J = 9 Hz, 1H), 7.03 (dd, J = 8.4, 2.4 Hz, 1H), 6.67 (d, J = 8.7 Hz, 1H), 6.48 (d, J = 16.2 Hz, 1H), 3.76 (s, 3H).

Cl

Cl

O

OCH3

Hapten T7

as coating hapten

O

NaOHMeOH H+

Compound 8 O

Cl

Cl

O

OCH3

OH

O

Scheme 7

Synthesis of (2E,4E)-5-(3-chloro-4-(2,4-dichlorophenoxy)phenyl)penta-2,4-dienoic acid (Hapten T8, BDH 382-70, Scheme 8):

A suspension of the aldehyde (Compound 15, 345 mg, 1.15 mmol), triethyl 4-phosphonocrotonate (350 mg, 1.27 mmol), LiOH.H2O (54 mg, 1.27 mmol), and activated 4 Å molecular sieve (1.9 g) in tetrahydrofuran and under a stream of nitrogen gas was refluxed overnight. The crude reaction mixture was filtered through silica gel, eluting with ethyl acetate. The mixture was concentrated. The residue was purified with silica gel using a mixture of ethyl acetate and hexane (1:10, v/v) to give Compound 18 (BDH-382-67) as a white solid (331 mg, yield: 73%). TLC (ethyl acetate/hexane, 1:5, v/v) Rf, 0.68. 1H NMR (300 MHz, chloroform-d) δ 7.58 (d, J = 1.8 Hz, 1H), 7.49 (d, J = 2.4 Hz, 1H), 7.45 (d, J = 5.4 Hz, 1H), 7.40 (dd, J = 9.6, 5.1 Hz, 1H), 7.28 (dd, J = 5.1, 2.7 Hz, 1H), 6.85 (d, J = 9.0 Hz, 1H), 6.80 (dd, J = 6.6, 1.8 Hz, 1H), 6.76 (d, J = 3.9 Hz), 6.00 (d, J = 15.0 Hz, 1H), 4.23 (q, J = 7.2 Hz, 2H), 1.32 (t, J = 6.9 Hz, 3H).

Compound 18 (250 mg, 0.63 mmol) was dissolved in 1,4-dioxane (5 mL) and hydrolyzed under alkaline conditions using LiOH.H2O (127 mg, 3.02 mmole) at 40 oC overnight. The reaction mixture was acidified with 6 N HCl and extracted with ethyl acetate. The organic layer was concentrated. The residue was recrystallized from a mixture of ethyl acetate and hexane to give Hapten T8 as a white solid (150 mg, yield: 65%). 1H NMR (600 MHz, DMSO-d6) δ 12.31 (s, 1H), 7.83 (dd, J = 28.0, 2.3 Hz, 1H), 7.52 (dd, J = 8.6, 2.1 Hz, 1H), 7.44 (dd, J = 8.8, 2.5 Hz, 1H), 7.31 (dd, J = 15.2, 10.9 Hz, 1H), 7.15 (dd, J = 15.6, 10.9 Hz, 1H), 7.06 - 7.01 (m, 1H ), 6.01 (d, J = 15.2 Hz, 1H).

Cl

Cl

O

Cl

CHO Cl

Cl

O

Cl

Compound 15Hapten T8

as coating hapten

OPOO

O

O

O

LiOH, THF,molecular sieve

LiOH.H2O,

Dioxane H+

Cl

Cl

O

Cl

OH

OCompound 18

O

Scheme 8

Synthesis of (E)-3-(3-chloro-4-(2,4-dichlorophenoxy)phenyl)-2-methylacrylic acid (Hapten T9, BDH 382-71,

Scheme 9): A suspension of the aldehyde (Compound 15, 500 mg, 1.67 mmol), triethyl 2-phosphonopropionate (438 mg, 1.84 mmol), LiOH.H2O (77 mg, 1.84 mmol) and activated 4 Å molecular sieve (2.76 g) in tetrahydrofuran and under a stream of nitrogen gas was refluxed overnight. The crude reaction mixture was filtered through silica gel, eluting with ethyl acetate. The mixture was concentrated. The residue was purified with silica gel using a mixture

S-8

of ethyl acetate and hexane (1:10, v/v) to give Compound 20 (BDH-382-69) as a white solid (556 mg, yield: 87%). TLC (ethyl acetate/hexane, 1:5, v/v) Rf, 0.75. 1H NMR (300 MHz, chloroform-d) δ 7.52 (d, J = 2.7 Hz, 1H), 7.49 (d, J = 2.7 Hz, 2H), 7.21 (dd, J = 8.4, 2.4 Hz, 2H), 6.87 (d, J = 8.7 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 4.27 (q, J = 7.2 Hz, 2H), 2.11 (d, J = 1.5 Hz, 3H), 1.35 (t, J = 6.9 Hz, 3H).

Compound 20 (400 mg, mmol) was dissolved in 1,4-dioxane (5 mL) and hydrolyzed under alkaline conditions using LiOH.H2O (127 mg, 3.02 mmole) at 40 oC overnight. The reaction mixture was acidified with 6 N HCl and extracted with ethyl acetate. The organic layer was concentrated. The residue was recrystallized from ethyl acetate and hexane to give Hapten T9 as a white solid (327 mg, yield: 88%). 1H NMR (600 MHz, DMSO-d6) δ 7.77 (ddd, J = 53.6, 6.1, 2.5 Hz, 1H), 7.55 (d, J = 4.9 Hz, 1H), 7.45 (ddq, J = 8.5, 5.9, 2.4 Hz, 1H), 7.14 – 7.00 (m, 1H), 2.03 (d, J = 3 Hz, 3H).

Scheme 9

Coupling methods

Sulfo-N-hydroxysuccinimide (NHS) Method. Hapten T1 was coupled covalently with the lysine moieties of the carrier proteins such as thyroglobulin. That is, each hapten (0.02 mmol) was dissolved in 1 mL of dimethylformamide (DMF) with sulfo-NHS (0.024 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 0.024 mmol). After the mixture was stirred overnight, the active ester was added slowly to a solution of thyroglobulin (25 mg of protein in 1 mL of 0.05 M borate buffer at pH 8) with vigorous stirring. The reaction mixture was stirred gently at 4 °C for 24 h to complete the conjugation. The conjugates were dialyzed against phosphate buffer saline (PBS) for 36 h with buffer changes every 12 h and stored at -20 oC until use.

N-Hydroxysuccinimide (NHS) Active Ester Method. The haptens T3, T4, and T5 containing a –COOH group were coupled covalently with the lysine moieties of carrier proteins such as thyroglobulin and bovine serum albumin according to the activated ester method. That is, each hapten (0.02 mmol) was dissolved in 0.2 mL of dry dimethylformamide with equimolar NHS and a 10% molar excess of dicyclohexylcarbodiimde. After the mixture was stirred overnight at 22 °C, the precipitated dicyclohexylurea was removed by filtration, and about 0.2 mL of the active ester was added slowly to a solution of the protein (25 mg of protein in 1 mL of 0.05 M borate buffer at pH 8) with vigorous stirring. The reaction mixture was stirred gently at 4 °C for 24 h to complete the conjugation and then dialyzed and stored as described above.

Mixed Anhydride Method. Haptens T7, T8 and T9 containing carboxylic acids were activated by the mixed anhydride method. Haptens (0.03 mmol) were dissolved in dry p-dioxane. Isobutyl chloroformate and tri-n-

butylamine were added in slight molar excess. The solution was stirred at room temperature for 30 min. Fifty milligrams of each protein (bovine serum albumin or LPH, Limulus polyphemus hemocyanin) was dissolved in 30 mL of 0.2 M borate buffer, pH 8. The protein solution was ice-cooled. To improve the solubility of the activated hapten in the aqueous protein solution, 2 mL of p-dioxane was added to the protein solution. The addition of p-

dioxane to the protein solution caused a slight cloudiness. The activated hapten solution was then added to the protein solution dropwise with stirring. Stirring was continued on ice for 0.5 – 1 h. To remove unreacted small molecules, the protein conjugates were precipitated with ice cold 100% ethanol. The precipitated protein was pelleted by centrifugation at 4 °C for 10 min, 4500g. The supernatant containing unreacted small molecules was decanted. The pellet was resuspended with cold ethanol three times, centrifuging between resuspensions. The

S-9

supernatants were discarded, and the pellet was resuspended in distilled water to a concentration of approximately 5 mg of protein/mL. All conjugates were assayed for protein content, aliquoted, and stored at -20 or -80 °C until use.

Diazotization Method. Hapten T2 was covalently conjugated to tyrosine moieties of the carrier protein. That is, 0.2 N sodium nitrite (0.5 mL) was added dropwise to a solution of the hapten (0.03 mmol) dissolved in a mixture of a few drops of ethanol and 0.2 N HCl (0.5 mL) until a positive starch iodide test was confirmed. The reaction vial was cooled with ice while the solution was stirred. Dimethylformamide (0.3 mL) was added and stirred for 10 min, and the solution was divided into two equal aliquots. One aliquot was added to a solution of thyroglobulin, the other to a solution of bovine serum albumin. The thyroglobulin (25 mg) and the bovine serum albumin (25 mg) were dissolved in 5 mL of ice-cold borate buffer (0.2 M, pH 8.9). The reaction mixtures were cooled in an ice bath and stirred continuously for 30 min. The pH of the yellow solutions was adjusted to 7.0 with 1 N NaOH. Each mixture was dialyzed and stored as described above. Instrumental Analysis of Water and Biosolid Samples

Analysis of Water samples: Water samples were extracted by solid phase extraction (SPE) using Oasis HLB cartridges (3 cc 60 mg, Waters, Milford, MA) as reported elsewhere (Charles et al., 2011). The HLB cartridges were first washed with 3 mL ethyl acetate, 3 mL methanol twice, and 3 mL 95:5 v/v water/methanol with 0.1% acetic acid. The 1 mL water samples were then loaded onto the cartridges. The samples were spiked with 100 µL 100 ng/mL internal standard (4-phenoxyphenol) and flowed through the sorbent by gravity. They were then washed with 3 mL 95:5 v/v water/methanol with 0.1% acetic acid twice and dried for 20 min with low vacuum. The triclosan was then eluted with 0.5 mL of methanol followed by 1.5 mL of ethyl acetate into tubes containing 6 µL of 30% glycerol in methanol as a trap solution. The volatile solvents were evaporated by using vacuum centrifugation until about 2 µL of trap solution remained in the tube. The residues were dissolved in 1 mL of methanol for LC/MS/MS analysis. All samples were extracted in triplicate. Analysis of Biosolid samples: Samples of dried biosolid powder (1.0 g) were extracted with a reflux column using methanol (15 mL) at a constant temperature (65°C) and with agitation, on an oil bath for two hours. The agitation process was maintained for another 30 min until the whole solution cooled down and the mixture was then filtered on a vacuum system using filter paper. Biosolid extract samples were purified with a solid phase extraction (SPE) before analysis by LC/MS-MS. Prior to extraction, 3 cc Waters Oasis®-HLB cartridges were washed with ethyl acetate (3 mL), methanol (2 × 3 mL), and 95:5 v/v water/methanol with 0.1% acetic acid (2 × 3mL). Biosolid extract (3 mL) was then loaded onto the cartridges. Cartridges were washed two times with 3 mL of 95:5 v/v water/methanol with 0.1% acetic acid. The aqueous plug was pulled through the SPE cartridges using high vacuum. The SPE cartridges were further dried with low vacuum about 30 min. SPE cartridges were eluted using 0.5 mL of methanol followed by 1.5 mL of ethyl acetate into 2 mL tubes containing 6 µL of 30% glycerol in methanol as a trap solution. The volatile solvents were removed using a Speed-Vac until only the trap solution of 2 µL of glycerol remained. The residues were reconstituted in 1 mL of methanol containing 10 ng/mL of internal standard (4-phenoxyphenol). The samples were then mixed on a vortex mixer for 1 min, transferred to autosampler vials, and stored at -20 °C until analysis. The liquid chromatography method followed that used by Ogunyoku and Young (2014). The system used for analysis was an Agilent 1200 SL liquid chromatography series (Agilent Corporation, Palo Alto, CA). The autosampler was kept at 4 °C. Liquid chromatography was performed on a reverse-phase Phenomenex® Luna 3µ C18 (2), 150 x 2.00 mm column. Mobile phase A was water with 0.1% glacial acetic acid. Mobile phase B consisted of acetonitrile with 0.1% glacial acetic acid. Gradient elution was performed at a flow rate of 300 µL/min. Chromatography was optimized to separate all analytes in 15 min, and the injection volume was 10 µL.

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The column was connected to a 4000 QTrap tandem mass spectrometer (Applied Biosystems Instrument Corporation, Foster City, CA) equipped with an electrospray source (Turbo V). The instrument was operated in negative multiple reaction monitoring (MRM) mode. TCS standard was infused into the mass spectrometer and MRM transitions and source parameters optimized. The MRM transitions used to quantify TCS and 4-phenoxyphenol were 289 Da-35 Da and 185 Da -108 Da, respectively. Peak integration and quantification was performed automatically using the Analyst® v1.6 software. The limit of detection (LOD) and limit of quantification (LOQ) for each of the compounds were determined as 3 and 10 times the signal to noise ratio, and were equaled to 7.67 ng/mL and 24.7 ng/mL, respectively. References Ahn, K. D., Gee, S. J., Tsai, H.-J., Bennett, D., Nishioka, M. G., Blum, A., Fishman, E., Hammock, B. D.

Immunoassay for monitoring environmental and human exposure to the polybrominated diphenyl ether BDE-47. Env. Sci. Technol. 2009, 43, 7784-7790.

Charles, R. L., Burgoyne, J. R., Mayr, M., Weldon, S. M., Hubner, N., Dong, H., Morisseau, C., Hammock, B. D., Landar, A., Eaton, P. Redox regulation of soluble epoxide hydrolase by 15-deoxy-∆-prostaglandin J2 controls coronary hypoxic vasodilation. Circ Res., 2011, 108, 324-334.

Freundlich, J. S., Anderson, J. W., Sarantakis, D., Shieh, H. M., Yu, M., Valderramos, J. C., Lucumi, E., Kuo, M., Jacobs, W. R. Jr., Fidock, D. A., Schiehser, G. A., Jacobus, D. P., Sacchettini, J. C. Synthesis, biological activity, and X-ray crystal structural analysis of diaryl ether inhibitors of malarial enoyl acyl carrier protein reductase. Part 1: 4'-substituted triclosan derivatives. Bioorg. Med. Chem. Lett. 2005, 15, 5247–5252.

Ogunyoku, T.A., Young, T.M. Removal of triclocarban and triclosan during municipal biosolid production. Water

Environ Res., 2014, 86, 197-203.