Identification and quantification of TBBPA and its metabolites in adult zebrafish by high resolution liquid chromatography tandem mass spectrometry

Microchemical Journal 154 (2020) 104566

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Identification and quantification of TBBPA and its metabolites in adult zebrafish by high resolution liquid chromatography tandem mass spectrometry

T

Fang Tana, Bin Lua, , Zengze Liub, Guangyu Chenb, Yanqun Liuc, Feifei Chengb, Yikai Zhoua, ⁎

⁎

a

Key Laboratory of Environment and Health, Ministry of Education & Ministry of Environmental Protection, and State Key Laboratory of Environmental Health (Incubating), School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, #13 Hangkong Road, Wuhan, Hubei 430030, China b School of Chemistry and Environmental Engineering, Jianghan University, Wuhan, Hubei 430056, China c School of Medicine, Jianghan University, Wuhan, Hubei 430056, China

ARTICLE INFO

ABSTRACT

Keywords: TBBPA HRMS Metabolite Identification Quantification

Tetrabromobisphenol A (TBBPA) is extensively used as brominated flame retardant. TBBPA and some of its metabolites had been identified as endocrine disrupter with potential health risk. Understanding the distribution characters of TBBPA and its metabolites in fish is essential to evaluate the potential impacts of TBBPA on aqueous ecosystem. This study developed a simple method to identify and quantify TBBPA and its metabolites simultaneously in tissues of alult zebrafish by ultra-high performance liquid chromatography couple with Orbitrap high resolution mass spectrometry(UHPLC-Orbitrap-HRMS). Six TBBPA metabolites were identified in liver, kidney, gill and muscle of adult zebrafish exposed to water containing different TBBPA levels, two of them, 1,3-dibromo-2‑methoxy‑5-vinylbenzene and 2,6-dibromo-4-nitrophenol were first detected. TBBPA and its metabolites were mainly distributed in the livers and kidneys of zebrafish. The levels of TBBPA and metabolites reached maximum on exposed day 3, then decreased gradually in liver and kidney. TBBPA oxidative cleavage products and TBBPA-monosulfate were the dominant metabolites. UHPLC-Orbitrap-HRMS provided an effective solution to study the distribution and metabolite characters of TBBPA in aquatic organisms.

1. Introduction Tetrabromobisphenol A (TBBPA) is widely used as an additive or reactive flame retardant in the production of polymers and printed circuit boards [1,2]. Because of its high lipophilicity and environmental stability, TBBPA could be easily transformed into the various aquatic environment [3, 4]and has been detected frequently in different aquatic organisms [5]. TBBPA could cause adverse effects in aquatic animals such as zebrafish [6], sea urchin [7], freshwater fish [8]. and rainbow trout [9]. by mimicking thyroid hormones (THs) due to its chemical similarity to THs [10]. Additionally, TBBPA also had estrogenic disrupt activity and reproductive toxicity in aquatic vertebrates at very low concentrations [11]. On the other hand, some TBBPA metabolites such as TBBPA-monomethylether (MM-TBBPA) and tribromobisphenol A(TriBBPA)[12,13] were identified to display higher side effects than TBBPA [14].It is important to study distribution and metabolism of TBBPA in aquatic organisms in order to understand the fate and potential impact of TBPPA on aquatic organisms.

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Current research was concentrated on the determination of TBBPA in aquatic animals [15,16], but the research on distribution and biotransformation of TBBPA and its metabolites was limited. In the results of in vitro TBBPA metabolism studies of liver microsomes and S9 fraction from crucian carp (Carassius carassius) [17],sulfate or glucuronide conjugates metabolites of TBBPA,which were identified as the main metabolites in mammal or amphibians [18,19], were not detected. The in vivo metabolism study of TBBPA in fish deserved additional investigations to clarify this difference between in vivo and in vitro. Another research on metabolism of TBBPA during embryonic and larval development in zebrafish could not provide TBBPA and its metabolites distribution in vivo data [20]. Mud carp(Cirrhinus molitorella) and northern snakehead(Ophicephalus argus) were collected to determine the concentrations of TBBPA in the different tissues [15], but TBBPA metabolites distribution has not been studied yet. Therefore, it is necessary to quantify not only TBBPA, but also identify and quantify more TBBPA metabolites in different tissues of aquatic animal to understand distribution and metabolism of TBBPA in vivo more

Corresponding authors. E-mail addresses: [email protected] (B. Lu), [email protected] (Y. Zhou).

https://doi.org/10.1016/j.microc.2019.104566 Received 9 October 2019; Received in revised form 20 December 2019; Accepted 21 December 2019 Available online 25 December 2019 0026-265X/ © 2019 Elsevier B.V. All rights reserved.

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comprehensively. At present, the main methods for the determination of chemical and its metabolites were shown in the Table 1. High performance liquid chromatography coupled with mass spectrometer (HPLC-MS) was the preferred method for quantitative analysis of TBBPA and its metabolites because of no requiring any derivatization in contrast to GC-based methods. In HPLC-MS methods, orbitrap high resolution MS(OrbitrapHRMS) was different from triple quadrupole MS (TQ-MS) in acquisition mode, using an extract ion chromatogram on the analyte theoretical m/ z with a narrow mass extraction window and mass resolution was very high .Moreover, the acquisition recorded virtually all ions present at the ionization source [21]. It was suited for non-target screening to qualitative determinations in MS and MS/MS modes [21], the scope of following quantitative analysis would increase since more unknown metabolites were identified. On the other hand, Orbitrap-HRMS were evaluated to be reliable and sensitive and comparable TQ-MS quantitative performance for its high resolution, accurate mass measurement and mass stability [22]. In this study, a simple and rapid method by UHPLCeOrbitrapHRMS was established for the first time to both qualitative and quantitative analysis of TBBPA and its metabolites in vivo. In this method, in addition to TBBPA quantitative analysis, non-targeted screening was used to identify more metabolites of TBBPA. Unknown metabolites could be identified by spectral characteristics of specific chromatographic peaks. On this basis, the metabolites were quantitatively analyzed simultaneously to provide distribution and transformation data of TBBPA in different tissues. The analysis efficiency in this method could be improved, but more importantly, the dataset obtained had a closer scientific integration for TBBPA metabolism research [21]. In the analytical procedures used for extraction of TBBPA and its metabolites from the biological medium, Soxhlet extraction was most commonly applied because of its robustness and low cost [15], but long processing time and large amount of solvent consumption limits its application . There were other several methods for extraction from samples including solid-phase extraction (SPE) and liquid–liquid extraction (LLE) [27,28]. These traditional extraction procedures are not very convenient and practical in determination on TBBPA and its metabolites. For these reasons, development of an alternative method for the extraction of TBBPA and its metabolites was very necessary. Zebrafish was widely used as bioindicator organism because of its low cost and convenient feeding. In addition, zebrafish, as one of a group of small fish, are easily exposed to particular environmental chemical in tank water in the laboratory, and exhibit measurable sensitivity to chemicals [29]. In this paper, zebrafish was selected as model vertebrate to identify and quantify TBBPA and its metabolites in aquatic animal tissues by UHPLCeOrbitrap-HRMS. The present study described the development of HRMS method for the identification and determination of TBBPA and its metabolites in tissues of adult zebrafish for the first time to our best knowledge. The method also included simple extraction of TBBPA from tissues of zebrafish for deproteinization and lipid removal. The purpose of this study was to apply this new analytical method to study TBBPA biotransformation and metabolism in aquatic animals, so as to provide some clues for the toxicity mechanism of TBBPA in aquatic animals.

2. Materials and methods 2.1. Materials TBBPA (99%) was obtained from Shanghai Aladdin Bio-Chem Technology Co., LTD. Methanol, dimethylsulphoxide (DMSO, 99%), nhexane and methylene dichloride were HPLC grade from Sinopharm Chemical Regent Co.Ltd. (China).All other solvents and reagents used in these experiments were of analytical grade or higher and were purchased from standard sources. AB Wild-type, (length (34 ± 5) mm; weight (350 ± 10) mg) three-month-old zebrafish obtained from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). 2.2. Fish culture and TBBPA exposure Zebrafish were acclimated in aquarium water at 28 ± 1 °Cwith a 12 h light: 12 h dark cycle for 10 days before the experiments. Air stones were placed to maintain oxygen saturation in the water. Fish were fed twice a day with live brine shrimp nauplii and checked daily for abnormal behavior, disease, and mortality. All animal experiments were approved by the Animal Experimental Ethics Committee of Huazhong University of Science and Technology. Zebrafish were randomly assigned to TBBPA exposure groups and control groups. In order to study the effects of TBBPA exposure concentration and exposure time on the method, based on toxicity test concentration general setting rules [30] and the toxicity index(96 h LC50) of TBBPA reported as 3 mg/L in zebrafish [31], the concentrations of TBBPA exposure were set as 0.5 mg/L(1/6 LC50), 1.0 mg/L (1/3 LC50) and 2.0 mg/L (2/3 LC50) . The stock solution of TBBPA was prepared in DMSO and the final DMSO concentration in water was 0. 2%. The volume of every tank was 3 L and the final volume of exposure solution was 2 L.The TBBPA contained water changed daily. Triplicate experiments were conducted for each treatment group. On exposure day 1, 3, 5, and 10, triplicate samples (each sample with ten zebrafish in a tank) in each treatment group were randomly removed and killed instantly. The weight and length of each individual fish were measured. The tissues of each fish was harvested, weighed accurately, snap frozen and stored at −20 °Cbefore extraction and analysis. Solvent control (0.2% DMSO) experiments were also conducted. 2.3. Sample preparation and extraction The sampled tissues were first homogenized in 600 μL methanol/ water solution (v/v = 4:1) for 5 min with a homogenizer (MY-10, Jinxin Scientific, China). Then the homogenized samples were centrifuged at 12,000 × g at 4 °Cfor 15 min. After centrifugation, 400 μL supernatant was vacuum-dried using a Speedvac concentrator (Thermo Scientific, USA).To the dried sample, 600 μL methylene dichloride / nhexane (v/v = 3:1)was added and then vortexed briefly and centrifuged (12,000 × g; 15 min at 4 °C). After centrifugation, 500 μL supernatant was dried down using Speedvac (Thermo Scientific,USA).The residue were reconstituted using 100 μL methanol, and then centrifuged at 12,000 × g at 4 °Cfor 15 min. Ultimately, the supernatant was collected and analyzed by UHPLCeOrbitrap-HRMS (Thermo Fisher Scientific, U.S.A).

Table 1 Methods for the determination of chemical and its metabolites. Method

Application

GC–MS HPLC-TQ-MS

derivatization needed for determination of TBBPA [23] widely used for quantification analysis [5], standard was required to optimize mass spectrometric parameters and acquisition mode could mask other valuable information [24] qualitative and quantitative analysis by high-resolution full-scan acquisition [25, 26]

HPLC-Orbitrap-HRMS

2

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2.4. Instrumental analysis

sensitivity was observed and space charge problems would occur resulting in a shift of m/z values [24]. So AGC target of 100,000, maximum injection time of 100 ms were set for full scan mode and AGC target of 10,000, maximum injection time of 50 ms were set for MS/MS mode. 5 ppm mass tolerance window was set to acquire adequate selectively.

TBBPA and its metabolites were analyzed by UHPLCeOrbitrap HRMS(Thermo, USA) with a Hypersil GOLD column (100 mm × 2.1 mm,1.9 μm, Thermo) at 35 °C. The injection volume was 20 μL. Separation was performed by gradient elution with water (Solvent A) and acetonitrile (Solvent B) at a flow rate of 0.3 mL/min using the following conditions: starting with A/B at 95:5 and switching to 5:95 at 8 min, holding for 2 min before returning to 95:5 and then keeping for 3 min. Analytes were detected in the ESI−ion mode with capillary temperature 320 °C, aux gas heater temperature 350 °Cand pray voltage 3200 V. Mass range in the full scan experiments was set at m/z 100–2000, whereas for MS/MS, it was set from m/z 200 to 800.The mass spectrometers were used with the following optimized parameters: resolution (70 000), AGC target (100 000), maximum injection time (100 ms) for full scan mode and resolution (17 500), AGC target (10 000), maximum injection time (50 ms) for MS/MS mode. The collision energy was 30ev to detect a high number of compounds. Default tolerances for RT(RT ± 0.5 min) and mass accuracy (mass tolerance ± 5 ppm) were provided a suitable detection of the compounds. Xcalibur Qual and Quan Browser software were used for the qualitative and quantitative calculation.

3.2. TBBPA metabolites identification The liver was chosen as a priority tissue to search for TBBPA metabolites because liver was the main metabolic organ in zebrafish [6]. On TBBPA exposure day 10, liver tissues of zebrafish in 2.0 mg/L treatment group was harvested, extracted and analyzed. Six TBBPA metabolites were identified by matching accurate masses and isotope patterns achieved by UHPLCeOrbitrap HRMS analysis and mass spectra interpretation on basis of literatures and mass spectral database (Fig. S2). They were 2,6-dibromo-4-[1-(3‑bromo‑4-hydroxyphenyl)−1-methylethyl]-phenol (TriBBPA) (M1), 1,3-dibromo2‑methoxy‑5-vinylbenzene (M2), 4-(2-hydroxyisopropyl)−2,6-dibromophenol(M3), 2,6-dibromo-4-nitrophenol (M4), and TBBPAmonosulfate (M6). Because mass spectrometry analysis did not provide exact positions of additional substituents on the aryl ring, 2,6-dibromo4-(1‑hydroxy‑2-methylpropan-2-yl)phenol or 2,6-dibromo-4-(2-hydroxyprpan-2-yl)−3-methylphenol (M5) should be considered as two methoxylated isomeric forms of 2,6-dibromo-4-isopropyl-phenol. The masses, chemical structures and the corresponding fragments of the six metabolites in the liver were listed in Table 2. None of these metabolites was detected in zebrafish of the control group. The parent compound TBBPA and metabolites M1 to M6 showed the same characteristic bromine isotope pattern, suggesting that these metabolites have bromine in their molecules. M1 (MW-H 464.8326) had characteristic bromine isotope patterns showing 3 bromines in its molecule and was identified as tribromobisphenol-A. M2(MW-H 290.8847) MS/MS fragment ions(m/z 78.9175) were consistent with mass spectra provided by Sun [34], was identified as 1,3-dibromo2‑methoxy‑5-vinylbenzene. M3(MW-H 308.8960) MS/MS fragment ions(m/z290.8851, 78.9176) were consistent with mass spectra provided by Liu [20], so M3 was identified as 44-(2-hydroxyisopropyl)−2,6-dibromophenol. The NIST mass spectral database was used as a reference to M4 MS/MS fragment ions (m/z236.9947, 186.9265, 78.9174).M4 was identified as 2,6-dibromo-4-nitrophenol . M5(MW-H 322.9112) MS/MS fragment ions(m/z290.8854, 78.9177) were consistent with mass spectra provided by Liu [20], had two methoxylated isomeric forms of 2,6-dibromo-4-isopropyl-phenol.There was no choice to determine and quantify individually these metabolites, and therefore they were quantified simultaneously.M6(MW-H 622.7028)was identified as the sulfated TBBPA, since SO3H accounted for the additional m/z 80.Metabolites M1, M3, M5 and M6 had been identified and detected in organisms [13,18,20,35], but M2 and M4 have not been detected in animals in the previous studies, only were found in the environment [34,36]. The discovery of these metabolites provided more possibilities for further study on the transformation and metabolism of TBBPA in aquatic animals.

2.5. Data statistical analysis All data were expressed as means ± standard deviation (S.D.).The levels of TBBPA and metabolites were prepared by one-way ANOVA with Turkey’ multiple comparison tests to evaluate the statistical significance of differences (p<0.05).For result handling analysis, concentrations below the LOQ were replaced with a value equal to LOQ divided by the square root of 2 for calculation. 2.6. Quality control of samples Standard solutions of TBBPA were included in each run in concentrations read from the calibration curve (low, middle, and high points) as control samples. 3. Results and discussion 3.1. .UHPLCeOrbitrap-HRMS optimization UHPLC conditions were optimized firstly to ensure a rapid and effective separation of the target compound. In the literature of Liu [32],methanol and water were applied as mobile phases, but acetonitrile as organic mobile solvent, showed better chromatographic signal of TBBPA in this study. Finally, gradient program, column temperature, flow rate, and injection volume were evaluated in terms of chromatographic selectivity and analysis time, TBBPA standard solutions was well analyzed within 13 min described in Section 2.4. The resolution factor was 1.8, capacity factor was 6.0, selectivity factor was 1.2, dead time was 1.34 min, theoretical plate number was more than 220,000. For MS parameters, negative ionization(ESI−) mode was used because the signal of TBBPA was better than in positive ionization (ESI+) mode since TBBPA had hydroxyl groups directly linked to benzene rings, which were relatively easy to lose hydrogen ions. The MS parameters were optimized with resolution, AGC target and maximum injection time. Selectivity and sensitivity needed to be taken into consideration simultaneously when setting the mass resolution. With the mass resolution increasing, mass accuracy(selectivity)will be better, but a too high resolution would significantly affect the sensitivity because of the reduced scanning speed [33]. In this study, a resolution of 70,000FWHM and 12,500 FWHM were chosen for full MS and MS/MS events, respectively. The automatic gain control (AGC) and maximum injection time setting were also found to be other important parameters. If the values were set too high, no significant improvement in

3.3. . Method development and validation 3.3.1. Recovery in the final procedure In order to detect TBBPA and its metabolites in biological samples accurately, sample pretreatment was necessary to remove interferences. In this study, a modified liquid extraction method using a series of solvents was established to reduce matrix effects [37]. First, precipitation of protein was one common strategy prior to analysis of TBBPA in tissues sample. The efficiency for deproteinization of the mixtures of methanol and water with different ratio (2:1, 3:1, 4:1) in blank tissue from zebrafish spiked with TBBPA standard solution was studied. The supernatant in the three precipitation tests was analyzed 3

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Table 2 TBBPA Metabolites and their structures detected by Orbitrap-HRMS.

by UHPLCeOrbitrap-HRMS to compare recovery (REC,%) of TBBPA. Methanol/water solution (v/v = 4:1) was the selective extraction step for deproteinization (Table S1.). Subsequently, according to literatures [5, 38], lipid removal efficiency of different ratios of dichloromethane and n-hexane (1:1, 2:1 and 3:1 (v/v)) was investigated to determine the optimal solvent mixture. The supernatant obtained from blank zebrafish tissue spiked with TBBPA standard solution after precipitation with methanol/water solution (v/v = 4:1) was vacuum-dried and used for lipid removal. The

final extraction was analyzed by UHPLCeOrbitrap-HRMS to compare recovery of TBBPA. The maximum lipid removal efficiency was attained by using the solvent mixture of 3:1 (v/v) dichloromethane and nhexane (Table S2).Therefore, methanol/water solution (v/v = 4:1) for deproteinization, and methylene dichloride / n-hexane (v/v = 3:1) for lipid removal, was the optimal sample pretreatment method. When spiked samples were pre-treated by this method, recoveries of TBBPA ranged from 64.5 to 70.5% with relative standard deviation (RSD) ranged from 8.5 to 13.6% (Table 3). The recoveries of TBBPA in 4

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sensitivity (RSD%<0.12%) and retention time(RSD%<2.3%) indicated that these parameters remain nearly un-affected by slight changes in the main chromatographic conditions. With UHPLCeOrbitrap HRMS method, TBBPA could be quantified according to the standard curve achieved from commercial standard. Without available standard, the amounts of M1 to M6 could be calculated by the ratio of peak area of the metabolite to the wet weight of sampled tissue. This method has been widely used in analyzing metabolites [35] because ionization efficiency of the metabolites in massspectrometry were similar [40-42]. Therefore, this method after development and validation by adding TBBPA standard solution to blank sample could be used for not only TBBPA quantification but also evaluating its metabolites transformation by the relative abundances in semiquantitative determination.

Table 3 The extraction recovery(n = 3), intra-day (n = 5), and inter-day (n = 5) precision and accuracy obtained in the quantification of TBBPA. Spiked Concentration

Recovery % RSD% (n = 5)

1 μg/L

64.5 (13.6) 65.8 (8.5) 70.5 (9.2)

10 μg/L 25 μg/L

Intra-day

Inter-day

Accuracy RSD% (n = 5)

Precision RSD% (n = 5)

Accuracy RSD% (n = 5)

Precision RSD% (n = 5)

11.2

10.2

10.2

7.9

8.7

5.8

6.5

7.2

6.3

5.9

5.4

6.1

this study were similar to other studies. For example, recoveries of TBBPA spiked human adipose tissue, muscle tissues from bull shark and Atlantic sharpnose shark were 65.5%, 62.8% and 65.5%, respectively [5]. High reproducibility was critical to ensure accurate determination of trace analytes in biological samples [39]. As the RSD of this sample pretreatment was lower than 13.6%, it had enough reproducibility for quantitative analysis of TBBPA.

3.4. Analysis of TBBPA and its metabolites in zebrafish tissues After the development stage of the method, the optimized analytical procedures described above were applied to the determination of TBBPA and its metabolites in different tissues of zebrafish on exposed day 1, 3, 5 and 10. The experiments results showed that concentrations of TBBPA in liver and kidney increased as the exposed levels of TBBPA increased. On the other hand, In 0.5 mg/L and 1.0 mg/L groups at each exposed time, no significant difference on TBBPA level was observed in gill and muscle(p>0.05). In 2.0 mg/L groups, the TBBPA concentration in gill increased obviously. On exposed day 3, the level of TBBPA in gill in 2.0 mg/L group was 9.9 times more than the level of TBBPA in gill in 0.5 mg/L group(Fig. 1). During the exposure period, concentrations of TBBPA in liver and kidney increased rapidly to maximum on exposed day 3, then decreased gradually in all groups(p<0.05). In 2 mg/L exposed group, the concentrations of TBBPA in liver and kidney on exposed day 10 were approximately 46.3% and 49.8% lower than those on day 3, respectively (Table.S3). The experiment results showed that TBBPA levels in zebrafish followed the order of liver>kidney>gill>muscle in all exposure groups(p<0.05) (Table. S4). Similar to TBBPA, The concentrations of M3, M5, M6 in liver and kidney increased as the exposed TBBPA concentrations increased (Fig. 2). The concentration of M2 in liver increased with the increase of TBBPA exposure concentration (p<0.05), but the changes were not significant in kidney, gill and muscle, possibly because of the lower concentration in these tissues. On the other hand, the concentrations of M4 in liver, kidney and gill decreased as the exposed concentrations increased (p<0.05). The concentrations of M4 in liver and kidney were higher in 0.5 mg/L than in other exposed groups. During exposed tests, similar distribution pattern of most TBBPA metabolites were observed in adult zebrafish, concentrations of most metabolites in liver and kidney reach the maximum on exposed day 3(Table S5). The concentrations of M2, M3, M5 and M6 in liver on exposed day 10 in 2.0 mg/L exposed group were approximately 56.7%, 48.9%, 50.1% and 52.3% lower than those on day 3 and the concentrations of M2, M3, M5 and M6 in kidney on exposed day 10 were approximately 52.8%, 24.1%, 54.2%, 38.1% lower than those on day 3, respectively. In 0.5 mg/L exposed group, the concentrations of M4 in liver and kidney on exposed day 10 were approximately 51.4% and 26.8% lower than those on day 3, respectively. Although the concentration of M1 in the kidney was below the LOQs during exposure

3.3.2. Linearity and sensitivity Under optimal procedural conditions, a blank sample of zebrafish tissue was spiked at six concentration levels of TBBPA in the range of 0.1 to 25 μg•L−1.Slope, intercept and determination coefficients (r2) were calculated by least square linear regression, plotting the areas of the chromatographic peaks for TBBPA versus concentrations(n = 5) (Fig.S1). TBBPA had good linearity in the range of 0.1–25μg•L − 1with r2=0.9906.The limits of quantification (LOQ, a signal to noise (S/N) ratio =10) was 3.2 ng/g (ww) and the limits of detection (LOD, a signal to noise(S/N) ratio =3) was 0.9 ng/g (ww), determined on wet tissue sample (n = 5). 3.3.3. Repeatability and accuracy Repeatability(intra-day) was determined by the analysis of five zebrafish tissue replicates fortified with the standard solution. Intermediate accuracy(inter-day) was carried out on 5 different days at the same concentration levels of TBBPA samples, which were prepared freshly. The RSD values were used as a measure for the precision (Table 3). As seen in Table 3, the method gave satisfactory precise results. 3.3.4. Matrix factor and carry factor The carry over effect was evaluated by analyzing a tissue sample spiked 1μg•L−1of TBBPA. It should be noted that blank tissue sample was analyzed and no peak of TBBPA was found in the corresponding chromatogram. Within the concentration range studied, the residue of the analyte in the needle must be removed before the next injection to avoid any interference. 3.3.5. Robustness The robustness of the method was evaluated by analyzing a standard solution of TBBPA(1μg•L−1,n=5),by making slight changes to the main chromatographic conditions. The variation of sensitivity (peak area) and retention time was calculated (Table 4). The low variability of Table 4 Evaluation of the robustness of TBBPA. Parameter The recommended condition Flow rate 0.28 mL/min Column temperature30 °C ⁎

Retention time(min) RSD%(n = 5)* 9.41 ± 0.08 10.02 ± 0.11 9.52 ± 0.12

All values were expressed as mean ± RSD. 5

Peak area RSD%(n = 5)* 903,069 ± 2.3 936,512 ± 1.5 884,512 ± 2.1

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Fig. 1. The concentrations of TBBPA in liver (a), kidney (b), gill (c) and muscle (d) in the 0.5,1.0,2.0 mg/L exposure groups. The concentrations were shown on the basis of the wet weight. The values were the means ± SD (n = 3).

Fig. 2. Distribution of TBBPA metabolites in tissues in 0.5, 1.0 and 2.0 mg/L groups. The relative amount of metabolite at per gram tissue was presented with peak area during the exposure period. The values were the means ± SD (n = 3). 6

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period, the concentrations of M1 in liver on exposed day 10 was approximately 47.1% lower than that on day 3 in 2 mg/L exposed group. Furthermore, similar to TBBPA, concentrations of M1, M2, M5 and M6 in the liver were the highest among all tissues(p<0.05)(Table S6). But the levels of M3 in liver were lower than those in kidney. M3 was a hydrophilic compound with hydroxyl groups, which may contribute to its rapid excretion to kindney to reduce its level in liver. On the exposed day 3 in 2.0 mg/L group, amount of metaboliteM3, M5, M6 in the liver of zebrafish accounted for 3.7%, 7.3%, 88.4% of the total amount of all metabolites, respectively. Thus, these three metabolites were the main TBBPA metabolites in zebrafish tissues during the exposure period. The absolute predominance of M6 in metabolites indicated that TBBPA was effectively metabolized to M6 by sulfur transferase. On the other hand, M3 and M5 were formed through oxidative cleavage near the central carbon of the molecule of TBBPA. The high levels of M3 and M5 indicated that TBBPA oxidative cleavage products were presumably other main metabolites in liver of zebrafish.

Science Foundation of China (Grant No. 21611130030 and 21277054).The authors thank Dr. Yong Liang and Dr. Yanmin Long at Hubei Key Laboratory of Environmental and Health Effects of Persistent Toxic Substances, Institute of Environment and Health, Jianghan University for their technical assistance and suggestions. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.microc.2019.104566. Appendix A. Supplementary data Supporting information for this article includes additional details of the recoveries of TBBPA (Tables S1–S2), the concentrations of TBBPA and its metabolites in different tissues in the exposure group during the exposure time (Tables S3–S6), Calibration line for TBBPA (Fig. S1) and Mass spectrum and secondary spectrum diagram of TBBPA and its metabolites (Fig. S2).

4. Conclusion To our knowledge, this manuscript reported the first study of TBBPA and its metabolites simultaneous identification and determination in fish different tissue samples by UHPLCeHRMS-Orbitrap. HRMSOrbitrap chromatography provided strong evidences of the presence of six TBBPA metabolites without resorting to the use of standards, two of these metabolites were first found in organisms. This analysis indicated that methanol/water and dichloromethane/n-hexane two-step extraction was a promising solvent extraction method and useful for exploratory analysis. In the validation of the method, the parameters repeatability, intermediate precision, matrix interference, quantitative limit, linearity, carry-over and robustness were evaluated. All parameters were satisfactory for determining concentration of TBBPA and metabolites relative abundances in tissues of adult zebrafish. The exposure experiment results showed TBBPA and its metabolites were mainly distributed in the livers and kidneys of zebrafish, and the levels of TBBPA and its metabolites in the livers and kidneys reached maximum on exposed day 3, then decreased gradually. TBBPA oxidative cleavage products and TBBPA-monosulfate were the dominant metabolites. These experimental data provided useful information on the biotransformation between TBBPA and its metabolites in fish subject to different environmental stresses. It would be helpful to study the potential metabolic pathways and the toxic mechanism of TBBPA exploration in aquatic animals.

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CRediT authorship contribution statement Fang Tan: Conceptualization, Methodology, Software, Writing original draft. Bin Lu: Supervision, Writing - review & editing. Zengze Liu: Software, Validation, Visualization. Guangyu Chen: Data curation, Formal analysis. Yanqun Liu: Investigation, Resources. Feifei Cheng: Software, Validation. Yikai Zhou: Funding acquisition, Project administration. Declaration of Competing Interest The authors declare that they have no conflict of interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Acknowledgements This work was supported by the National Basic Research Program of China (973 Program) (2015CB352102) and the National Natural 7

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