MS

Journal Pre-proof Determination of 19 anthelmintics in environmental water and sediment using an optimized PLE and SPE method coupled with UHPLC-MS/MS

Yiwen Li, Zhiwei Gan, Yunxiang Liu, Sibei Chen, Shijun Su, Sanglan Ding, Ngoc Han Tran, Xi Chen, Zhimin Long PII:

S0048-9697(20)31027-5

DOI:

https://doi.org/10.1016/j.scitotenv.2020.137516

Reference:

STOTEN 137516

To appear in:

Science of the Total Environment

Received date:

18 December 2019

Revised date:

20 February 2020

Accepted date:

22 February 2020

Please cite this article as: Y. Li, Z. Gan, Y. Liu, et al., Determination of 19 anthelmintics in environmental water and sediment using an optimized PLE and SPE method coupled with UHPLC-MS/MS, Science of the Total Environment (2020), https://doi.org/10.1016/ j.scitotenv.2020.137516

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© 2020 Published by Elsevier.

Journal Pre-proof

Determination of 19 anthelmintics in environmental water and sediment using an optimized PLE and SPE method coupled with UHPLC-MS/MS Yiwen Li1, Zhiwei Gan*1, Yunxiang Liu1, Sibei Chen1, Shijun Su1, Sanglan Ding1, Ngoc Han Tran2, Xi Chen3, Zhimin Long3 1. College of Architecture and Environment, Sichuan University, Chengdu, 610065, China

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2. Duy Tan University, Da Nang, 550000, Viet Nam.

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3. SCIEX, Analytical Instrument Trading Co., Shanghai, 200335, China

ABSTRACT

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A sensitive quantification method using pressurized liquid extraction (PLE) and solid phase

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extraction (SPE) coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed for determination of 19 anthelmintic drugs (ADs)

Hexahydropyrazines,

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belonging to seven structural groups (Benzimidazoles, Diphenylsulfides, Imidazothiazoles, Macrocylic

lactones,

Salicylanilides,

Tetrahydropyrimidines)

in

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environmental water and sediment samples. Eleven SPE cartridges, sample pH, elution solvents were tested to determine the optimal conditions for extraction. Among these investigated SPE

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

types, the best recoveries for 19 target ADs were obtained from Oasis HLB cartridge with 37– 102%, 45–103%, 37–88%, 28–82% and 31–90% for spiked river water, tap water, rainwater, wastewater, and sediment respectively (with RSD<15%), except for closantel. The 19 ADs were separated within 10 min by a BEH C18 column and monitored in both positive and negative ions modes with switching electrospray ionization source. The cross-talk interferences were solved by identification of secondary mass spectrum of substances through MRM-IDA-EPI scanning using Qtrap. These interference peaks could be efficiently eliminated by setting MRM segments or using Qtrap to obtain tertiary fragmented information. The developed methods were satisfactory 1

Journal Pre-proof in terms of linearity, accuracy, and precision, and used eight isotopically labeled compounds as internal standards to correct matrix effects. Method quantification limit (MQL) for 19 ADs was below 1.1 ng/L, 0.4 ng/L, 5.4 ng/L and 2.3 ng/g for river water, tap water, wastewater, and sediment, respectively. The validated method was successfully used to investigate the occurrence of anthelmintics in water and sediment samples from Chengdu, China. All ADs were detected in environment with the concentrations at ng/L level.

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Keywords: anthelmintics; PLE; SPE; UHPLC-MS/MS; water; sediment

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1 Introduction

Over last decades, pharmaceuticals and personal care products are regarded as emerging

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contaminants in environment (Silva et al., 2011; Kolpin, 2002; Le et al., 2018; Petrie et al., 2015;

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Utrilla et al., 2013; Tran et al., 2019; Tran et al., 2018). Anthelmintic drugs (ADs), an important class of veterinary pharmaceuticals, are widely used for the prevention and treatment of parasitic

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infections in agriculture, aquaculture, and in some cases for human infections (Horton, 2000; Horvat et al., 2012; Waller, 1997). In addition, some ADs have also been used as pre or

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post-harvest fungicides for preventing fungi which might affect field crops, stored fruit and

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vegetables (Danaher et al., 2007; Schirra, 2002). Although ecotoxicological studies on aquatic species and human have shown that ADs are relatively safe to take in appropriate dose for animals and the side effects are predictable for human (Boxal et al., 2003; Dusi et al., 2005; Lumar et al., 2002; Wagil et al., 2015a; Yoshimura, 2005), the ecological effect of ADs on the environment remains unknown. Due to their high consumption, ADs potential influences on the aquatic and terrestrial environment have drawn much attention and become a new research interest. Human drugs are usually treated by wastewater treatment plants (WWTPs) before discharging into the aquatic environment (Le et al., 2018; Tran et al., 2016b), but veterinary drugs such as 2

Journal Pre-proof ADs and their metabolites always enter the environment directly through urine and feces from animals, or slurry and manure spreading in animal treatment processes and aquaculture activities (Boxal et al., 2003; Zrncic et al., 2014). To date, most studies on ADs focused on their levels in food products such as milk, eggs and fish (Chen et al., 2011; Dasenaki and Thomaidis, 2015; Kinsella et al., 2009; Silva et al., 2017; Whelan et al., 2010; Zhang et al., 2011), and human biological samples (plasma, blood, urine and feces) (Chhonker et al., 2018; Danaher et al., 2007;

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Msagati and Nindi, 2006; Pandya et al., 2017). Only few studies were reported the occurrence and fate of some ADs in the environment (Cacho et al., 2009; Kim et al., 2017; Krogh et al., 2008;

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Wagil et al., 2015b), in which ADs were detected at significantly lower concentrations compared

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to other pharmaceuticals (Bartelt-Hunt et al., 2009; Bottoni et al., 2010; Cerqueira et al., 2014;

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Charuaud et al., 2019; Löffler and Ternes, 2003). Due to its own corresponding parasites, multiple ADs are usually used in combination. Thus, it is crucial to develop a highly reliable analytical

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method for achieving simultaneous quantification of various ADs.

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Previous analytical method for the determination of one or two broad spectrum (i.e. macrocyclic lactones, benzimidazoles) of ADs in solid and aqueous environment (Cacho et al.,

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2009; Islam et al., 2013; Krogh et al., 2008; Mutavdzic Pavlovic et al., 2012; Santaladchaiyakit

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and Srijaranai, 2012; Tejada-Casado et al., 2016; Van de Steene et al., 2006) are summarized in Table S1 in the Supplementary materials (SM). In most cases, ADs was monitored with other multi-class pharmaceuticals in environmental matrices (Dasenaki and Thomaidis, 2015; Petrovic et al., 2014; Peysson and Vulliet, 2013; Sim et al., 2013). The low concentration and particular physicochemical properties of ADs were the factors that make it difficult to analyze a variety of ADs simultaneously (Tran et al., 2016a). Whelan (2010) presented a modified QuEChERS extraction method for analysis of 38 ADs in milk samples with UHPLC-MS/MS, which filled the gap on simultaneous detection of ADs in biological samples. The first ultrasensitive method for detecting 10 ADs in surface water samples was developed by Zrncic (2014), using SPE method followed by UHPLC coupled to quadrupole linear ion trap mass spectrometry. In the case of solid 3

Journal Pre-proof samples, PLE was recommended for obtaining more concentrated extracts compared with other classical extraction methods (i.e. microwave assisted extraction, liquid-liquid extraction and supercritical fluid extraction) (Drljača et al., 2016). However, some drawbacks found on previous methods for environmental screening could be improved, such as sensitivity, extraction efficiency and severe matrix effects. Therefore, the objective of this study was to develop a fast, sensitive and selective analytical

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method for simultaneous determination of 19 ADs belonging to 7 classes using UHPLC-MS/MS. Pretreatment approach involving SPE and PLE for water and sediment samples were established

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and optimized. Internal standard calibration including 8 labeled internal standards was used to

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obtain more accurate quantification results. For method validation, precision, accuracy, recovery

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and other parameters were evaluated. Matrix effects of the analytes were assessed by post-extraction spike method and corrected efficiently by internal standards. The method was

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validated and applied on determination of 19 target compounds in sediment, river water, tap water,

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rainwater and wastewater from Chengdu, a big city with extensive agriculture and livestock activity in central-western China. These occurrences data provide a better understanding of ADs

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2 Experimental

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fate, behavior and potential risk in environment.

2.1 Chemicals

These 19 target analytes in this study are high consumption compounds in agriculture and livestock, including: 1) Benzimidazoles: Albendazole (ALB), Ricobendazole (RIC), Fenbendazole (FEN), Flubendazole (FLU), Mebendazole (MEB), Oxfendazole (OXF), Thiabendazole (THI). 2) Diphenylsulfides: Bithionol (BIT), Febantel (FEB). 3) Imidazothiazoles: Levamisole (LEV). 4) Hexahydropyrazines: Diethylcarbamazine (DIE). 4

Journal Pre-proof 5) Macrocylic lactones: Abamectin (ABA), Doramectin (DOR), Eprinomectin (EPR), Ivermectin (IVE), Moxidectin (MOX). 6) Salicylanilides: Closantel (CLO). 7) Tetrahydropyrimidines: Morantel (MOR); Pyrantel (PYR). The

ADs

standards

were

purchased

from Dr.Ehrenstorfer

Thiabendazole-D4(THI-D4),

Levamisole-D5(LEV-D5),

Diethylcarbamazine-D3(DIE-D3),

Germany).

Fenbendazole-D3(FEN-D3), Closantel-C6(CLO-13C6),

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Albendazole-D7(ALB-D7),

(Augsburg,

Roxithromycin-D7(ROXI-D7) and Febantel-D6 (FEB-D6), used as internal standards (ISs), were

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obtained from Dr.Ehrenstorfer (Augsburg, Germany), Toronto Research Chemicals (North York,

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Ontario, Canada), and Witega Laboratorien (Berlin, Germany), respectively. The structures of

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these ADs are listed in Table S2 in the SM.

In addition, HPLC-grade acetonitrile, acetone and methanol were obtained from Fisher

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Scientific (Waltham, Massachusetts, USA). Other chemical reagents like HPLC-grade ammonium

used throughout the study.

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2.2 Standard solutions

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acetate and formic acid was purchased from CNW (Düsseldorf, Germany). Milli-Q water was

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All standards of ADs and their isotopically labeled compounds were dissolved in methanol and stored in the dark at -20◦C. Due to different solubility, primary stock solutions were prepared with different concentrations, from 0.1 mg/mL for FLU and MEB, 0.2 mg/mL for RIC and FEN, 0.5 mg/mL for ALB, CLO and PYR, and to 1 mg/mL for the other 12 ADs. A mixture of all target ADs (10 μg/mL) was prepared in methanol containing 50% Milli-Q water (v/v), used for standard curves and sample fortification. The mixed working standard (50 ng/mL) was prepared in Milli-Q water before SPE experiment. A mixed IS solution including 8 analytes were prepared in methanol/water (1:1, v/v) with concentration of 10 μg/mL and stored at -20◦C. 5

Journal Pre-proof 2.3 Sampling and sample pretreatment The surface water samples and sediment samples were collected in Jiangan River and Ming Yuan Lake, and the tap water samples and rain water samples were obtained at public places in Chengdu, China. The wastewater sampling sites were the influent and effluent of two different WWTPs. Samples were collected using plastic bottles and stored in the dark at 4◦C until analysis. No preservation agent was added. Before the SPE test, all water samples were filtered at room

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temperature using 0.45 μm cellulose nitrate membrane filters (Whatman, Maidstone, UK). The

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pH of samples was adjusted by hydrochloric acid solution (0.1 M) until the desired pH (5-7) was reached. Sediment were air dried and sieved through a 2 mm sieve to obtain homogeneous

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samples before further analysis

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2.4 Pressurized liquid extraction (PLE)

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The sediment samples were extracted using a Speed Extractor E-916 (Buchi Labortechnik AG, Flawil, Switzerland). Approximately 10 g of dry sediment was put into a 50 mL extraction cell

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filled with sea sand. Both the bottom and top of the extraction cell were placed with cellulose filters, in order to prevent samples from penetrating the metal filter. The whole PLE procedure

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time was 45 mins. Firstly, preheating samples to testing temperatures (40◦C, 70◦C and 100◦C) for

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5 mins, and then using different solvent 1) methanol, 2）methanol/acetone (1:1, v/v), 3) methanol/water (1:1,v/v) and 4) acetone/water (1:1,v/v) to extract compounds for 5 mins in 3 cycles. The pressure was set up at 100 bar, and purge time was chosen as 3 mins. The extracts were evaporated and diluted with Milli-Q water before SPE.

2.5 Solid phase extraction (SPE) The optimization of the SPE procedure was conducted by adding mixed working standards into Milli-Q water. Eleven disposable SPE cartridges were tested, including Waters Oasis WAX (3 mL, 60 mg), Oasis MAX (3 mL,60 mg), Oasis MCX (3 mL, 60 mg), Oasis WCX (3 mL, 60 mg), 6

Journal Pre-proof Oasis HLB (3 mL, 60 mg) (Milford, Massachusetts, USA), and CNW Poly-Sery MAX (6 mL, 150 mg), CNW Poly-Sery MCX (6 mL, 150 mg), CNW Poly-Sery WAX (6 mL, 150 mg), CNW Poly-Sery HLB (6 mL, 500 mg) (Düsseldorf, Germany), Strata-X (3 mL, 60 mg) (Phenomenex, California, USA) and Chromabond ® HR-X (6 mL, 500 mg) (Macherey Nagel, Düren, Germany). Wash buffer solutions including Milli-Q water containing methanol, formic acid (FA) and ammonia hydroxide (NH4OH) were used to optimize SPE procedure. Methanol, methanol

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containing NH4OH, methanol containing FA, acetonitrile and acetone were tested as elution solvents.

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The main framework of this optimized SPE method for 19 ADs was laid out in Table S3.

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2.5.1 SPE Test round 1

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All SPE cartridges were tested according to the general approach. Briefly, 6 mL methanol was

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added onto sorbent for condition followed by 6 mL Milli-Q water at same pH value with samples at a flow rate of approximately 1 mL/min. Then, 5 mL working standard solution were loaded and

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passed through cartridge. 6 mL Milli-Q water containing 5% methanol was used as wash buffer solution to remove impurities for HLB, Strata-X and HR-X cartridges, and 6 mL Milli-Q water

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containing 2% FA and 5% NH4OH were used to wash MCX and WAX, MAX and WCX

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cartridges, respectively. The HLB, Strata-X and HR-X cartridges were eluted with 6 mL methanol, MCX and WAX cartridges were eluted with 4 mL methanol followed by 2 mL methanol containing 5% NH4OH, MAX and WCX cartridges were eluted with 4 mL methanol and 2 mL methanol containing 2% FA. Finally, extracts were evaporated under a nitrogen stream at 40◦C and reconstituted in 0.5 mL of a mixture of methanol and water (1:1, v/v).

2.5.2 SPE Test round 2 The cartridges (Waters Oasis HLB) with best performance in the test round 1 was chosen for further optimization. Considering the pH influence on extraction efficiency, pH value of working standard solution was adjusted to 3 to 10 using hydrochloric acid solution (0.1 M) and sodium 7

Journal Pre-proof hydroxide solution (0.1 M). The elution of 19 ADs from SPE cartridge were compared by eluting with 1) methanol; 2) methanol containing 5% NH4OH; 3) methanol containing 5% FA; 4) acetonitrile; 5) acetone. The elution steps were repeated for three times (3×2 mL) to result 6 mL extracts. The final extracts were evaporated gently to approximately dry with nitrogen and re-dissolved in 0.5 mL methanol/water (1:1, v/v). Before UHPLC-MS/MS analysis, a mixed IS solution (20 ng/mL) was added into the extracts. The sample breakthrough was studied by

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analyzing ADs in eluents collected after extraction with SPE cartridges.

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2.6 UHPLC-ESI-MS/MS analysis

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UHPLC-MS/MS analysis was performed using SCIEX Exion LC system coupled with SCIEX Triple Quad 4500 Qtrap (Framingham, Massachusetts, USA). The electrospray ionization (ESI)

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interface operated simultaneously in the positive (+) and negative (-) modes. The parameters for

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the MS system were set as following: curtain gas 30 psi, ion spray voltage 5000 V and -4500 V, source temperature 250°C and ion source gases (GS1 and GS2) were set to 65 psi and 35 psi,

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respectively. The optimal MS conditions for each target ADs were determined by injecting standard solution (50 ng/mL) directly to the mass spectrometer. MultiQuant 3.0.3 software was

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used for data acquisition and processing, and Analyst 1.7 software was used for data analysis. The

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related parameters for multiple-reaction monitoring (MRM) acquisition, retention time (RT), declustering potential (DP), collision energy (CE) and collision cell exit potential (CXP) for each compound are presented in Table 1. The separation of ADs was achieved using a BEH C18 column (2.1×100 mm, 1.7 μm, Waters, Milford, Massachusetts, USA). The column temperature was 45°C. The flow rate of gradient elution was 0.4 mL/min. The mobile phase gradient started with 10% B (methanol), increasing to 100% B at 3.5 min and held for 3.5 min. Then eluent B was lower back to initial conditions over 0.5 min. Finally, the column was equilibrated for 2.5 min. The injection volume was set to 5 μL.

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Journal Pre-proof 2.7 Method validation The linearity of the method was evaluated by external calibration. Ten-point external calibration curves were established with the addition of 8 internal standards, ranging from 0.05 to 200 ng/mL. Real river water, tap water and sediment samples were used for matrix matched calibrations. The extracts of real samples were spiked with 1, 5, 10, 50, 100 ng/mL ADs. The instrumental detection limits (IDLs) and quantification limits (IQLs) for 19 ADs were

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estimated from signal-to-noise ratio (S/N) of the minimum measured concentrations of calibration, which were calculated to 3 and 10 times of the S/N, respectively. The IQLs was also validated by

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standards with corresponding calculation concentration (Fig. S1).

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The method detection limits (MDLs) and method quantification limits (MQLs) were

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determined from 250 mL of spiked river water, tap water (1 ng/mL) and 10 g sediment (1 ng/g) as the lowest observable concentration of the analyte giving a S/N ratio of 3 and 10, respectively.

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For samples contaminated initially by ADs, MDLs and MQLs were estimated by determining the

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S/N ratios of the minimum detectable amounts and extrapolating to S/N values of 3 and 10, respectively (Gan et al., 2013).

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Both intra-day and inter-day precision for the instrument and analytical method were evaluated

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by calculating the relative standard deviation (RSD) of three replicate measurement. The precisions for the instrument were assessed by injecting standard solutions and post-spiked river water samples consecutively at different concentration in one day and in three different days, respectively. The repeatability and reproducibility of the analytical method was determined by analyzing 250 mL river water samples spiked before extraction at 5 and 10 ng/mL 19 ADs in one day and in three days. Blank samples were analyzed to evaluate possible contamination caused by instrument or interference of other samples. Absolute recovery (RE) and matrix effects (ME) used a post-extortion spiking method described by Matuszewski (2003) to evaluate. The measured background-subtracted peak area of the spiked sample prior to SPE, non-spiked sample and sample spiked after extraction were used 9

Journal Pre-proof to calculate absolute and relative RE, and ME values, detail equations are showed in Section S1 in the SM. For water and sediment samples, recovery and matrix effects were determined in two samples from two source in triplicate. The detailed preparing process for spiked samples are presented in Table S4.

3 Result and discussion 3.1 Optimization of UHPLC-MS/MS analysis

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The mass spectrum of the ADs was obtained by injecting standards at concentration of 50

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ng/mL. Most compounds achieved the highest sensitivity in positive ionization mode, except for

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BIT and CLO, which obtained the maximum sensitivity in negative ionization mode. The ion [M+NH4]+ was chosen for ABA, DOR and IVE, while the base peak for other ADs were [M+H]+

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or [M-H]-. For all analytes, identification was based on two MRM transitions and quantification

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was depended on the most intense product ion. The detailed MRM information are presented in Table 1. The MRM channel of PYR contained peaks at retention time (Rt) 2.60 min and 2.76 min,

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while those for MOR were 3.00 min and 3.15 min. The MRM-IDA-EPI scanning results of PYR

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and MOR were given in Fig. S2 and Fig. S3. Two different peaks (A and B) of PYR (207.1-150.1/136.1) and MOR (221.2-123.0/150.2) showed exactly same mass spectrum when

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using MRM-IDA-EPI scan with 4500 Qtrap, indicating that these peaks are most likely to have same molecular weight. Based on the chemical structure of PYR, it is inferred that it has stereoisomers. The result was consistent with Whelan studies who also found two peaks for MOR, confirming that MOR photoisomerization occurred in the presence of ultraviolet light (Whelan et al., 2010). In addition, the cross-talk interferences were observed among THI-D4 and LEV, PYR (Fig. S4). LEV and PYR showed an inference peak at Rt 3.27 min, while THI-D4 had a peak with the same retention time. Through MRM-IDA-EPI scanning, it was found that precursor ions and product ions for LEV and PYR was 205.1-178.2/123.0 and 207.1-136.1/150.1. The mass spectrum of interference analytes (205.1-178.1/134.2 and 207.1-180.1/136.1) was partially 10

Journal Pre-proof different compared by 4500 Qtrap (Fig. S2 and Fig. S5), suggesting that these peaks was not same substances. The interference peak could be sufficiently separated using chromatographic procedure and eliminated by setting up time segments MRM. Another way to solve the influence of interference peak is through Qtrap to obtain tertiary fragmented information (Fig. S6). The ESI-MS/MS parameters for monitoring the 19 ADs and 8 ISs are optimized and showed in Table 1.

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For LC separation, a Waters Acquity UHPLC BEH C18 column (2.1×100 mm, 1.7 μm) which was recommended in previous studies was examined (Zrncic et al., 2014). Compared to other

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columns, it contributed to diminish broad peaks and have less retardation for 19 ADs. Methanol,

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acetonitrile, and a mixture of methanol/acetonitrile (1:1, v/v) as organic phase, Milli-Q water with

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FA, ammonium formate (HCOONH4), ammonium acetate and acetic acid at various concentrations as aqueous phase were tested to optimize the MS response. Comparing mentioned

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three organic phases, methanol resulted in the best signal intensity for most ADs and ISs,

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especially for some ADs (i.e. macrocyclic lactones) with lower detection sensitivity. Adding FA and HCOONH4 in mobile phase simultaneously had a more significant contribution to gain good

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peaks and improve detection sensitivity than other compositions, which might provide more

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ammonium adducts and protons during ionization process. In particular, the response of macrocyclic lactones increased dramatically when HCOONH4 was added into aqueous phase instead of ammonium acetate. Therefore, Milli-Q water containing 5 mM HCOONH4 and 0.05% FA (mobile phase A) and methanol (mobile phase B) were chosen as mobile phase. The Fig. 1 gives a representative chromatographic separation of a mixed standard (20 ng/mL) using BEH C18 column (2.1×100 mm, 1.7 μm) based on the complete and optimized multi-residue UHPLC-MS/MS methodology.

3.2 Optimization of SPE In Test round 1, eleven SPE cartridges categorized into polymeric sorbents (HLB, Strata-X,

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Journal Pre-proof HR-X), polymeric sorbents with cation-exchange (MCX, WCX), and polymeric sorbents with anion-exchange (MAX, WAX) were tested using the method described in Section 2.6. The results are shown in Table S5. Polymeric sorbents were widely applied to pharmaceuticals extraction from aqueous sample in previous studies like HLB sorbents (Lin et al., 2005; Petrovic et al., 2014; Xue et al., 2015), due to its poly-divinylbenzene-co-N-vinylpyrrolidone copolymer, which provides hydrophilic-lipophilic retention characteristic. The results turned out that the highest

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average recovery of 19 ADs was yield from HLB cartridges (67%), followed by Strata-X cartridges (60%) and HR-X cartridges (59%). For basic compounds, MCX and WCX sorbents are

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a good choice because of strong cation exchange with acid groups, and these sorbents type indeed

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obtain relatively satisfactory recovery (>50%) for most compounds. However, reversed-phase

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part of MCX sorbents did not retain some chemical compounds which tended to be acids or neutrals well, such as FEB and BIT. The recovery of FEB was found lower than 20% when using

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MCX and WCX sorbents, which increased to 79% with HLB sorbents. The results indicated the

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characteristics of cartridges and the first pKa values of compounds were both crucial factors for extraction. As expected, MAX and WAX sorbents containing anion-exchanger did not perform

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with a satisfactory recovery for all ADs. Especially for LEV, DIE, PYR and MOR, the recoveries

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showed a significant decline when using these sorbents. But for CLO, it obtained the best recovery (52%) when using WAX sorbents. Oasis HLB cartridges performed better stability throughout the extraction process than CNW HLB cartridges and other cartridges, thus, this cartridge were further optimized in Test round 2 regarding wash buffer solutions, elution solvents, sample pH and volume. In the first round experiment, the Milli-Q water containing 5% methanol and methanol was used as wash buffers and elution solvents, while other wash buffers (Milli-Q water containing 2% FA, 2% NH4OH) and elution solvents composition (4 mL methanol + 2 mL methanol containing 2% FA or 2% NH4OH (v/v); 6 mL methanol + 2 mL acetonitrile; 6 mL methanol + 2 mL acetone) were evaluated in Test round 2. The effect of the composition of wash buffer and elution solvent on the 12

Journal Pre-proof recoveries are presented in Table S6. The results showed that adding acids or bases into wash buffer solution and elution solvent could improve the recoveries for majority of ADs to some degree, while the additives also caused some interferences in matrix effect. The recoveries of macrocylic lactones, which were lower than 50% in previous experiment, increased by 5-10% when using acetone as elution solvents. It suggested that acetone could elute more hydrophobic ADs than methanol. In conclusion, Milli-Q water containing 5% methanol and the composition (6

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mL methanol + 2 mL acetone) were chosen as wash buffer solution and elution solvent, respectively, and the overall recovery was around 72% (except for CLO).

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For ionogenic compounds like ADs (Gan et al., 2013), sample pH was a critical factor on

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recoveries. Thus, samples were adjusted in a range of pH 3 to 10 (Table S7). Compared to acidic

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and neutral buffer system, the recoveries of ADs decreased obviously at alkaline pH conditions. For CLO, the best recovery was obtained at acidified solvent (more than 60%), but it was not to

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be recommended due to instability of CLO in strong acidic conditions. Moreover, DIE only yield

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recovery of less than 30% in acidic condition, which was likely prone to be ionized. As a satisfactory recovery (Oasis HLB: 81%) was obtained at pH 5 to 7 for all ADs, except for

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macrocylic lactones (around 55%) and CLO (around 55%), the further investigation was

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performed using HLB cartridges under weak acidic and neutral buffer system. This result is consistent with the conclusion reported by Zrncic (2014) that the optimal recoveries for investigated ADs was obtained at pH 7. In general, for benzimidazoles and macrocylic lactones, the recoveries (ranging from 89 to 104% and 43 to 62%) were similar or little higher than those of other methods presented in Table S1, while the recovery of LEV increased from 43 to 89% in our studies (Zrncic et al., 2014). Concentrating is a common method for analyzing compounds at trace level in environmental samples. However, a large volume could increase the matrix effects and cause severe breakthrough. Overall recoveries for ADs decreased obviously when the sample volume

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Journal Pre-proof increasing from 250 to 500 mL (Table S8), especially for CLO. Therefore, 250 mL sample volume was chosen. Breakthrough experiments were conducted since relatively lower recoveries was obtained for macrocyclic lactones and CLO. Less than 7% of the macrocyclic lactones were detected in elutes after extraction with Oasis HLB sorbents. It indicated the low recoveries for macrocyclic lactones might due to their strong adsorption on the sorbent and the unsuitability of the elution solvent and

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wash buffer, but the recoveries were stable for environmental screening. For CLO, some losses (around 18% of analytes) was found in elutes, which could be explained by the Oasis HLB

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sorbents have a poor adsorption capacity for CLO. However, with stable recovery and using its

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isotopic standard as internal standard, CLO could be accurate quantitative. Finally, the optimized

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method was completed and applied in real samples.

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3.3 Optimization of PLE

In this study, pure methanol, mixtures of methanol/acetone (1:1, v/v), methanol/water (1:1, v/v)

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and acetone/water (1:1, v/v) were tested for extraction of spiked sediment samples at 40°C and 100 bar. The results (Table S9) showed that pure methanol and methanol/acetone (1:1, v/v)

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obtained the poorest average recoveries for 19 ADs, more than half of ADs had a recovery of less

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than 50%, which suggested pure organic solvent was not an appropriate choice. The methanol/water combinations achieved best recoveries with the lowest recoveries of BIT reaching up to 76%. Compared to methanol/water, matrix effect was more severe under acetone/water condition for sediment samples. Three extraction temperature (40°C, 70°C and 100°C) were chosen to evaluate its effect on recovery. Table S9 illustrated that the temperature of 70°C leads to the best extractability for all 19 ADs, with an average recovery of 93%. In order to investigate the possible influence on recovery and matrix effect with increased amounts in testing cell, the sample sizes (5 g, 10 g) were compared. The difference between these recoveries (64% and 66%) and matrix effects (80% and 72%) demonstrated sample sizes just had 14

Journal Pre-proof a limited effect, which could be ignored. Therefore, considering the low concentration of ADs might be in the sediment, 10 g is a better choice for environmental screening. Finally, 10 g sediment samples with ADs spiked at 50 ng/g were used for PLE experiment, with extraction temperature of 70°C, pressure of 100 bar, pre-heating for 5 min and repeating for 3 cycles. The extracts were evaporated and diluted to 250 mL using Milli-Q water and passed through SPE cartridges for clean-up. The recoveries and matrix effects of the whole extraction

of

process for sediment samples are presented in Fig. 2 and Fig. 3. Except for macrocylic lactones and CLO, all ADs achieved satisfactory recoveries in excess of 50%. However, relatively poorer

ro

recoveries were obtained in sediment than those in surface water for most compounds, might be

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owing to more complex matrices and extraction step for sediment pretreatment.

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3.4 Method validation

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3.4.1 Linearity, method detection limit (MDL) and quantification limit (MQL) The external calibration curve of 19 ADs was established with a good linearity and RSD of R2

na

were lower than 15 in all cases (Table 2). Macrocyclic lactones and tetrahydropyrimidines (PYR and MOR) showed lower sensitivity than the other groups. The IDLs and IQLs values of the 19

ur

ADs ranged from 0.6 to 18 ng/L and 2 to 60 ng/L, respectively (Table 2). The MQL values of 19

Jo

ADs in river water, tap water and sediment samples were from 0.09 to 1.07 ng/L, 0.02 to 0.36 ng/L and 0.03 to 2.26 ng/g, respectively. In terms of sensitivity, this method shows a better or similar performance for benzimidazole and avermectin class than other methods (ranging from 0.13 to 0.37 ng/L, 2.4 to 12 ng/L, respectively) (Zrncic et al., 2014; Krogh et al., 2008). For all analytes, the sensitivities were high enough for measurement of environmental samples with preconcentration.

3.4.2 Precision and accuracy Standard solutions (3 ng/mL and 6 ng/mL) and river water samples post-spiked with concentration at 5 ng/mL and 10 ng/mL were used to evaluate the precisions of triplicate 15

Journal Pre-proof experiment of instrument intra-day and inter-day. The repeatability and reproducibility of analytical method were determined by the analysis of three replicates of river water samples spiked before extraction in one day and three days, and the results are presented in Table S10. Overall, the precision of the method was acceptable with relative standard deviation (RSD) ranging from 1 to 15%. The different matrix components in samples collected from diverse sources might be a reason leading to one outlier with RSD higher than 15%.

of

3.4.3 Recovery (RE) and matrix effect (ME)

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During electrospray ionization mass spectrometry analysis, matrix components in environmental samples might interfere with the target compounds, resulting in ion suppression or

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enhancement. The MEs for 250 mL of river water, tap water, waste water, rainwater, and 10 g

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sediment from two different sources are presented in Fig. 2, and detailed data are summarized in

lP

Table S11. Ion suppression occurred in all matrices for all 19 ADs. A severe negative between 37 to 81% was verified with WWTP influent, while it varied from 52 to 84% in WWTP effluent. The

na

ME could either be compensated by internal standards (ISs), or reduced by concentrating diluted raw wastewater samples. The high level of ADs found in wastewater samples (Section 3.6)

ur

allowed us to dilute the raw samples without any quantification influence. For river water

Jo

samples, MEs were between 56 to 102%, with a satisfactory relative standard deviations (RSDs) value for the majority of ADs. Ion suppression were found weaker in the tap water and rainwater samples, with a range of 69-108% and 60-87% for MEs. As expected, MEs in sediments were also with a broad range from 48 to 90%. The common ion suppression phenomena could be explained by the co-elution existing between impurities in environmental samples and ADs with low retention in chromatographic column (Huang et al., 2006; Loos et al., 2009; Shao et al., 2009). Due to the MEs, the recovery in real samples was lower than that in Milli-Q water. It could be seen from Fig. 3 and Table S12 (except for CLO), the RE values of ADs in river water, tap water

16

Journal Pre-proof and rain water were from 37 to 103%. Consistently, the RE values decreased significantly in wastewater samples, with ranging from 28 to 75 %, and 31 to 82% in WWTP influent and effluent samples, respectively. For sediment samples, the RE values of 19 ADs were from 38 to 83%, which were relatively lower than those in water samples. In all cases, the precision of RE was acceptable, with RSD less than 15.

3.5 Optimization of quantification using deuterium-labeled internal standards

of

Mixed ISs were adopted and added into both standards and real samples before extraction, in order to minimize the matrix interferences, ion suppression and instrument fluctuation, as well as

ro

loss in pre-concentration on targets quantification. Seven 2H and a

13

C-isotope-labeled ISs,

-p

including ALB-D7 for ALB and RIC, FEN-D3 for FEN, OXF, FLU, MEB, THI-D4 for THI,

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FEB-D6 for FEB and BIT, DIE-D3 for DIE, LEV-D5 for LEV, MOR and PYR, CLO-13C6 for

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CLO, ROXI-D7 for five macrocylic lactones were chosen to compensate matrix effects for the quantification of 19 ADs. The results of recoveries and matrix effects for the ISs in the

na

investigated water and sediment samples are presented in Table 3. Because isotope-labeled analogues were not found in ADs for macrocyclic lactones group, the IS for the same type of

ur

antibiotic compounds was used, e.g. ROXI-D7. Its extraction recovery and matrix effects in

Jo

environmental samples was similar with target compounds. Thus, it is a suitable IS for macrocyclic lactones group in future studies. For CLO, due to the correction of CLO-13C6, the obtained concentration results could also be used as a reference for understanding CLO occurrence in environment. Internal calibration curves were constructed by plotting compounds concentration levels against the ratio of ADs peak area to corresponding ISs peak area. The accuracy of quantification has been further optimized by spiking ISs into above mentioned real environmental samples. As expected, the analog ISs could not completely compensate matric effect, because of the discrepancies between different chemical structures. But it has met the requirements (75-125%)

17

Journal Pre-proof recommended by AOAC guideline (Thompson et al., 2002). Therefore, the analytical method was valid and applicable by adding ISs.

3.6 Application to environmental water samples Fifteen water samples and three sediment samples were analyzed using the developed method, and the results are shown in Table 4. A broad-spectrum of ADs occurred in environmental samples, with the detection frequencies up to 63%. The highest mean concentration of target

of

compounds was detected in influent waste water (38.1 ng/L), followed by effluent waste water

ro

(22.9 ng/L), river water (6.0 ng/L), rain water (4.5 ng/L) and tap water (0.1 ng/L). In WWTP influent samples, 18 out of 19 target ADs were found at concentrations ranging from below LOQ

-p

to 432.8 ng/L. The removal efficiencies of 18 ADs in the WWTP ranged from -68 to 90%. Some

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ADs with low removal efficiencies (i.e. RIC and PYR) could discharge into the aquatic

lP

environment via WWTP effluent, which might be a contamination source of surface water. The most abundant AD found in surface water was MEB, followed by DIE and PYR. Compared with

na

previous studies, the mean concentration of LEV in river water in China (8.0 ng/L) were little higher than those in Europe (6.0 ng/L) (Zrncic et al., 2014). For sediment samples, targets

ur

compounds with rather high logKow values and sorption coefficients were defined at relatively

Jo

high concentration, such as BIT and CLO. They were found higher level in sediment than those in paired river samples.

4 Conclusions An analytical method involving pressurized liquid extraction (PLE) and solid phase extraction (SPE) as pretreatment process coupled with UHPLC-MS/MS was developed for the simultaneous determination of 19 anthelmintic drugs in environmental water and sediment samples. The proposed method shows some differences and novelty with respect to previous analytical approaches mainly focused in its more comprehensive monitoring categories, effective treatment procedure, and lower limits of detection for anthelmintic drugs were obtained at ng/L level, which 18

Journal Pre-proof were sensitive enough for analysis trace compounds in the environment. Environmental samples at neutral and weakly acidic conditions using Oasis HLB sorbents with methanol followed by acetone as eluent yielded satisfactory recoveries for most anthelmintic drugs. The PLE method stands for 10 g sediment samples, extraction solvent using methanol/water (1:1, v/v) and extraction conditions including temperature of 70°C, pressure of 100 bar, pre-heating for 5 min and repeating for 3 cycles. The matrix effects were evaluated and compensated with eight internal

of

standards. This is the first study that reports the concentrations of 19 anthelmintic drugs in real environmental samples including river water, tap water, rain water, wastewater and sediment in

-p

ro

China.

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Declaration of Competing Interest

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The authors declare no competing financial interest.

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Acknowledgement

Reference

ur

This study was supported by the Natural Science Foundation of China (No. 21607108).

Jo

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Toxicity of anthelmintic drugs (fenbendazole and flubendazole) to aquatic organisms. Environ. Sci. Pollut. Res. Int. 2015a; 22: 2566-73. Wagil M, Maszkowska J, Bialk-Bielinska A, Stepnowski P, Kumirska J. A comprehensive approach to the determination of two benzimidazoles in environmental samples. Chemosphere 2015b; 119 Suppl: S35-41. Waller PJ. Anthelmintic resistance. Veterinary Parasitology 1997; 72: 391-412. Whelan M, Kinsella B, Furey A, Moloney M, Cantwell H, Lehotay SJ, et al. Determination of anthelmintic

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Journal Pre-proof Yoshimura HE, Y. S. Acute toxicity to freshwater organisms of antiparasitic drugs for veterinary use. Environ. Toxicol. 2005; 20: 60-6. Zhang X, Xu H, Zhang H, Guo Y, Dai Z, Chen X. Simultaneous determination of albendazole and its metabolites in fish muscle tissue by stable isotope dilution ultra-performance liquid chromatography tandem mass spectrometry. Anal. Bioanal. Chem. 2011; 401: 727-34. Zrncic M, Gros M, Babic S, Kastelan-Macan M, Barcelo D, Petrovic M. Analysis of anthelmintics in surface water by ultra high performance liquid chromatography coupled to quadrupole

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re

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linear ion trap tandem mass spectrometry. Chemosphere 2014; 99: 224-32.

24

Journal Pre-proof

Retention time (min)

Precurso r ion (m/z)

Daughter ions

DP (vol ts)

EP (vol ts)

CE (vol ts)

CX P (vol ts)

Corresponding internal standard

ALB

positiv e

4.18

266

234

100

10

29

12

ALB-d7

190.9

100

10

44

12

ALB-d 7

positiv e

241.1

108

10

28

12

234

108

10

45

12

240

85

10

19

10

208

85

10

34

10

268

120

10

30

10

158.9

120

10

46

10

268.1

120

10

30

10

159

120

10

46

10

110

10

45

11

OXF

positiv e

FLU

positiv e

MEB

positiv e positiv e

THI-d4

positiv e

FEB

positiv e

FEB-d 6

positiv e

LEV

positiv e

LEV-d 5

positiv e

DIE

DIE-d3

4.32

303.2

3.55

316.3

3.97

314.2

3.89

3.19

296.1

3.19

Jo

THI

300

positiv e positiv

4.35

4.35

2.5

2.5

2.28

2.28

ro

positiv e

4.32

-p

FEN-d 3

282

159 191.1

110

10

28

11

282

120

10

33

13

122.9

120

10

48

13

263.9

120

10

30

8

105.1

120

10

43

8

175

80

10

35

8

130.9

80

10

45

8

179.1

80

10

31

10

135.2

80

10

46

10

383.1

95

6

27

5

415

95

6

19

5

383.1

95

6

27

5

418.1

95

6

19

5

178

85

10

29

11

123

85

10

39

11

183.1

95

10

30

10

128.1

95

10

38

11

100.2

60

10

20

10

127.1

60

10

20

10

100.1

70

10

21

10

lP

FEN

positiv e

3.33

na

positiv e

273.2

202.1

ur

RIC

4.18

206

447.1

453.1

205.1

210.1

200.2

203

of

Name

ESI mode

re

Table 1 Optimized mass spectrometer parameters for determination of anthelmintics

25

ALB-d7

FEN-d3

FEN-d3

FEN-d3

FEN-d3

THI-d4

FEB-d6

LEV-d5

DIE-d3

Precurso r ion (m/z)

Daughter ions

DP (vol ts)

EP (vol ts)

CE (vol ts)

CX P (vol ts)

130.1

70

10

19

10

305.1

85

10

34

14

567.4

85

10

21

14

331.2

80

10

35

13

593.3

80

10

22

13

569.3

85

10

22

9

307.1

85

10

35

9

186.1

110

10

25

13

154

110

10

62

13

528.3

95

ro

Name

Retention time (min)

ESI mode

10

13

10

120

10

31

9

re

Journal Pre-proof

120

10

41

9

95

10

18

10

150

85

10

38

10

136.1

85

10

41

10

122.9

90

10

44

10

164.2

90

10

36

10

160.8

-60

-10

-32

-10

191.9

-60 -12 0 -12 0 -12 0 -12 0

-11

-34

-10

-12

-97

-8

-13

-51

-8

-10

-90

-10

-10

-50

-10

Corresponding internal standard

DOR

positiv e

IVE

positiv e

EPR

positiv e

MOX

positiv e

5.07

890.5

5.25

916.5

5.5

892.5

4.97

914.5

5.34

640.4

-p

ABA

positiv e

687.6 ROXID7

positiv e

PYR

positiv e

4.39

845.6

158.2

positiv e

BIT

negati ve

CLO-1 3C6

negati ve

352.9

ur

negati ve

4.91

221.1

Jo

CLO

3.02

na

MOR

207

lP

498.3 2.54

4.87

660.7

4.87

666.6

of

e

126.9 344.8 126.7 350.7

26

ROXI-D7

ROXI-D7

ROXI-D7

ROXI-D7

ROXI-D7

LEV-d5

LEV-d5

FEB-d6

CLO-13C6

Journal Pre-proof Table 2 Method validation results Tap water External Analyte

calibration R2

IDLs

IQLs

(ng/L)

(ng/L)

River water

Sediment

MDLs

MQLs

MDLs

MQLs

MDLs

MQLs

(ng/L)

(ng/L)

(ng/L)

(ng/L)

(ng/g)

(ng/g)

0.9992

1.8

6

0.02

0.06

0.03

0.09

0.02

0.07

RIC

0.9984

1.8

6

0.03

0.09

0.06

0.20

0.06

0.20

FEN

0.9996

0.6

2

0.01

0.02

0.03

0.08

0.01

0.03

OXF

0.9994

1.8

6

0.02

0.05

0.05

0.14

0.03

0.11

FLU

0.9995

6

20

0.04

0.12

0.09

0.30

0.20

0.61

MEB

0.9995

1.8

6

0.03

0.09

0.06

0.19

0.05

0.16

THI

0.9995

1.8

6

0.02

0.07

0.05

0.16

0.04

0.12

FEB

0.9991

0.6

2

0.01

0.03

0.03

0.09

0.02

0.06

LEV

0.9991

1.8

6

0.03

0.08

0.05

0.18

0.05

0.16

0.08

0.25

0.15

0.46

0.23

0.70

0.34

1.03

0.15

0.22

0.65

0.33

1.01

0.10

0.09

0.29

0.11

0.34

0.9997

6

20

0.04

0.13

0.9992

18

60

0.06

0.18

DOR

0.9983

6

20

0.05

IVE

0.9998

1.8

6

0.03

EPR

0.9992

18

60

0.09

0.27

0.24

0.75

0.38

1.14

MOX

0.9995

6

20

0.03

0.10

0.12

0.35

0.27

0.82

PYR

0.9995

18

60

0.12

0.36

0.35

1.07

0.75

2.26

MOR

0.9994

18

60

0.05

0.14

0.22

0.67

0.33

1.01

BIT

0.9991

6

20

0.04

0.11

0.09

0.26

0.11

0.35

CLO

0.9991

1.8

0.01

0.03

0.03

0.10

0.02

0.06

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ALB

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Journal Pre-proof

Table 3 The results of recoveries and matrix effects for the 8 internal standards in the investigated water and sediment samples (n=3, RSD) (%) Analyte

River water

Tap water

Rain water

WWTP influent

WWTP effluent

Sediment

(Spiking level)

(5 ng/mL)

(5 ng/mL)

(5 ng/mL)

(100 ng/mL)

(100 ng/mL)

(50 ng/g)

RE

ME

RE

ME

RE

ME

RE

ME

RE

ME

RE

ME

ALB-D7

90±5.0

85±4.3

88±6.9

81±1.4

80±9.0

82±2.0

65±5.0

66±4.8

70±3.9

79±5.3

75±4.6

74±7.1

FEN-D3

83±5.5

80±6.3

86±8.1

86±2.1

79±4.4

78±3.1

58±7.1

59±3.9

69±5.4

69±4.7

74±8.6

73±7.0

THI-D4

90±3.4

83±2.2

89±2.6

81±6.8

93±2.7

77±5.1

66±5.4

55±3.3

78±1.7

72±4.0

80±8.9

76±9.7

f o

o r p

FEB-D6

81±4.9

81±4.6

74±4.6

75±1.9

75±2.5

77±5.0

45±3.4

58±1.7

57±8.0

63±7.3

77±4.4

64±2.3

LEV-D5

88±6.0

91±3.0

89±2.9

90±4.2

87±7.9

88±2.6

80±2.8

86±8.2

83±2.8

84±3.9

82±6.8

87±2.7

DIE-D3

88±2.6

87±6.2

87±8.2

89±3.8

84±4.2

83±6.5

68±6.1

74±6.6

74±5.0

80±2.0

73±4.2

77±5.3

ROXI-D7

68±3.8

85±6.2

70±3.7

80±6.2

76±3.7

80±4.5

54±4.2

64±4.1

63±4.3

78±3.0

53±3.8

73±6.2

·CLO-13C6

16±7.7

59±8.6

19±5.3

60±3.3

15±4.6

9±5.0

49±5.0

13±2.9

53±4.5

10±1.0

51±3.3

l a

r P

e

50±3.1

n r u

o J

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Journal Pre-proof Table 4 Occurrence of ADS in various water samples, Chengdu, China RIC

FE N

OX F

FL U

ME B

THI

FE AB DO MO PY MO BI CL LEV DIE IVE EPR B A R X R R T O

6.1

8.8

2.8

4.0

3.5

45.7

1.7

0.6 7.5 6.3 NQ

River-2

3.2

4.5

9.6

NQ

10. 2

13.3

2.4

1.3 4.1 20.7 NQ

River-3

2.9

7.5

4.5

3.8

7.0

40.1

4.3

0.3 12.4 2.6

Rain-1

2.7

ND

4.5

NQ

0.7

14.8

3.4

1.2 5.4 6.2 ND

Rain-2

ND

2.9

1.1

1.9

0.9

65.7

2.3

1.0 2.1 2.4 ND

Rain-3

9.7

2.9

1.3

4.0

0.6

29.3

1.2 ND 1.3 9.2 1.7 1.2 2.2 2.4 ND 21.9 2.3

Tap-1

2.6

1.8

ND

ND

ND

ND

ND ND ND ND ND

Tap-2

1.8

ND

ND

0.6

ND

ND

ND ND ND 0.1 ND ND ND ND ND ND ND ND ND

Tap-3

0.7

NQ

ND

NQ

Influent-1

40.7 8

66.4

7.9

NQ

Influent-2

53.7

72.4

19. 7

7.7

Influent-3

33.7

50.9

16. 3

Effluent-1

6.2

112. 1

Effluent-2

5.3

Effluent-3

NQ

103. 3

1.7

Sediment -1

2.6

2.8

2.8

Sediment -2

1.9

30.1

Sediment -3

5.1

3.1

98.9

ND

ND ND ND 11.4 7.5 3.8 5.5 ND ND ND 10.9 4.8 2.9 0.6

10.4 14.6 13.9 7.72 NQ 5.0 ND 6.9 4.3 8 4 8 ND ND ND

of

ND ND

ro

-p

re

ND

ND

ND ND ND ND ND

ND

ND

ND

4.9 2.9 1.4

ND ND ND 5.4 1.4 2.4 3.6 13. 9.2 4

ND ND ND ND ND ND ND

ND ND ND ND ND ND ND

lP

ND

223. 5

21.2

15. 22.2 22.0 10.5 13.9 22.9 7.7 ND 7.6 15.8 3.0 0.7 7

7.6

432. 8

121. 40. 145. 104. NQ 3.5 ND ND ND 5.8 ND 2.6 5.5 4 9 4 7

10. 3

183. 7

90.1

10. 113. 93.3 NQ NQ ND ND ND 14.6 7.9 9.6 4.4 1 4

9.6

98.5

66.6

35. 32.2 5.0 NQ ND NQ ND 7.4 8.3 7.5 1.0 1 ND

9.2

35.4

171. 40. 125. 50.2 ND ND NQ ND 4.2 ND 2.4 0.9 0 4 2 ND

NQ

10.3

82.2

17.2

8.3

12.2

2.4

9.9

9.9

13. 6

23.5

4.2

26. 140. ND 13.4 NQ ND ND 5.6 0.7 7.7 2.4 6 2 ND

8.8

7.8

5.4

29.9

0.8

12. NQ 18. 0.5 5.1 NQ NQ ND NQ 9.1 2 ND ND 4

6.8

na

River-1

NQ

ur

ALB

8.5

Jo

Concentrati on (ng/L)

3.5

ND 6.7

ND

ND：not detected；NQ: Detected but below LOQ

29

20. 40.4 30.2 ND NQ NQ 7.8 ND 6.2 7.4 6.0 0.4 3 ND

1.2 52.3 ND

ND

ND

ND

ND

1 ND 2.3 7.0 7.3

Journal Pre-proof Fig. 1 Chromatographic separation of 19 analytes at concentration of 20 ug/L. Fig. 2 The results of matrix effects for the tested ADs in the investigated water samples and sediment samples

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Fig. 3 The results of recoveries for the tested ADs in the investigated water samples and sediment samples

30

Journal Pre-proof CRediT authorship contribution statement

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Yiwen Li: Writing - original draft, Investigation, Formal analysis, Methodology. Zhiwei Gan: Writing - review & editing, Supervision, Funding acquisition, Conceptualization. Yunxiang Liu: Investigation. Sibei Chen: Investigation. Shijun Su: Writing - review & editing, Supervision. Sanglan Ding: Writing - review & editing, Supervision. Ngoc Han Tran: Writing - review & editing, Supervision. Xi Chen: Technical support. Zhimin Long: Technical support.

31

Journal Pre-proof

Graphical abstract

Highlights The pressurized liquid extraction (PLE) and solid phase extraction (SPE) method for anthelmintics was optimized.



19 anthelmintics were simultaneously detected by ultra-high performance liquid chromatography-tandem mass spectrometry.



This approach was used successfully to analyze ng/L levels of different classes of anthelmintics in environment.



The method was applied to analysis of river, tap, rain, waste water samples and sediment samples.



The cross-talk interferences were identified by Qtrap through MRM-IDA-EPI scanning.

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Figure 1

Figure 2

Figure 3