Determination of human metabolites of chlorinated phosphorous flame retardants in wastewater by N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide-derivatization and gas chromatography-high resolution mass spectrometry

Accepted Manuscript Title: Determination of human metabolites of chlorinated phosphorous flame retardants in wastewater by N-tertbutyldimethylsilyl-N-methyltrifluoroacetamide-derivatization and gas chromatography-high resolution mass spectrometry Authors: Ver´ onica Castro, Rosario Rodil, Jos´ e Benito Quintana, Rafael Cela, Laura S´ anchez-Fern´ andez, Iria Gonz´ alez-Mari˜ no PII: S0021-9673(19)30624-7 DOI: https://doi.org/10.1016/j.chroma.2019.06.015 Reference: CHROMA 360294 To appear in: Journal of Chromatography A Received date: 15 April 2019 Revised date: 4 June 2019 Accepted date: 7 June 2019 Please cite this article as: Castro V, Rodil R, Quintana JB, Cela R, S´ anchezFern´ andez L, Gonz´ alez-Mari˜ no I, Determination of human metabolites of chlorinated phosphorous flame retardants in wastewater by N-tert-butyldimethylsilylN-methyltrifluoroacetamide-derivatization and gas chromatography-high resolution mass spectrometry, Journal of Chromatography A (2019), https://doi.org/10.1016/j.chroma.2019.06.015 This is a PDF file of an unedited manuscript that has been accepted for publication. 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This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 1 Determination of human metabolites of chlorinated phosphorous flame retardants in wastewater by N-tert-butyldimethylsilyl-Nmethyltrifluoroacetamide-derivatization and gas chromatography-high resolution mass spectrometry Verónica Castro, Rosario Rodil, José Benito Quintana*, Rafael Cela, Laura SánchezFernández, Iria González-Mariño* Department of Analytical Chemistry, Nutrition and Food Sciences, IIAA – Institute for Food Analysis and Research, Universidade de Santiago de Compostela, Constantino Candeira S/N, 15782 – Santiago de Compostela, Spain. *Corresponding author: Iria González Mariño Phone: +34 881 816035 E-mail: [email protected] *Co-corresponding author: José Benito Quintana Phone: +34 881 816035 E-mail: [email protected] ACCEPTED MANUSCRIPT 2 Highlights:  A method to determine organophosphate (OP) diesters in sewage has been developed  OP diesters are rapidly silylated showing characteristic mass spectra  First GC-based method for OP diesters in wastewater extracts  Bis(chloropropyl) phosphate has been quantified at levels > 60 ng/L for the first time Non-intrusive approach for assessing human exposure to OP Abstract The analysis of wastewater for the determination of human biomarkers of exposure (human metabolites) is a non-intrusive, economic and complementary alternative to the analysis of urine in the monitoring of human exposure to chemicals of concern. This study provides the first gas chromatography-based method for the determination of three metabolites of chlorinated organophosphorous flame retardants (OPFRs): (bis(2chloroethyl) phosphate, bis(chloropropyl) phosphate and bis(1,3-dichloro-2-propyl) phosphate) in wastewater. A solid-phase extraction procedure based on the use of mixed-mode reversed-phase weak anion exchange sorbents was optimized including a fractionated elution of OPFRs and their metabolites. Analytes derivatization was investigated by comparing two silylating reagents, N-tert-butyldimethylsilyl-Nmethyltrifluoroacetamide and N-methyl-N-(trimethylsilyl)trifluoroacetamide, the first one providing better results. Determination was performed by gas chromatography-high resolution mass spectrometry with a quadrupole-time-of-flight system (GC-QTOF) in order to improve selectivity. Furthermore, the use of GC-QTOF combined with the specific ion obtained from silylated metabolites (m/z 154.9924) can be exploited to screen for other phosphate ester metabolites. Under final conditions, the overall method performance was satisfactory, affording method detection limits ranging from 1.1 to 4.6 ng/L, percentages of recovery from 90% to 110%, and relative standard deviations ACCEPTED MANUSCRIPT 3 below 13%. The analysis of composite raw wastewater samples collected over 24 h in the NW of Spain allowed to quantify, for the first time in this matrix, the metabolite bis(chloropropyl) phosphate at levels over 60 ng/L. Keywords: phosphorous flame retardants and plasticizers; metabolites; wastewater; human exposure; silylation; gas chromatography-high resolution mass spectrometry 1. INTRODUCTION Organophosphate esters, particularly triesters, are high-production-volume chemicals generally used as flame retardants and plasticizers (FRs) in a wide range of consumer goods, such as plastics, foams, paints, resins, textiles, electronics and furniture [1, 2]. Non-chlorinated alkyl phosphates are most commonly used as plasticizers, whereas chlorinated derivatives are used as flame retardants, receiving altogether the combined name of organophosphorous FRs (OPFRs) [2]. These compounds are employed as additives, i.e. mixed into the material but not chemically bonded to it, what facilitates their release into the surrounding environment and their further distribution into other environmental compartments [1]. They have been quantified in indoor air and dust [35], outdoor air, suspended particulate matter, sewage, surface and ground water, biota, soils and sediments [2, 6-8]. Their ubiquity and special high concentrations in indoor atmospheres entail a continuous exposure to OPFRs by the majority of the population, mainly via dermal absorption, ingestion of dust or contaminated food and inhalation of the most volatile species [9-13], tris(2-chloroethyl) phosphate (TCEP) and tris(chloropropyl) phosphate (TCPP, a technical mixture of four isomers differing in the alkyl chain) [5, 8, 14-16]. Harmful effects of this exposure include dermatitis (TCPP and 1,3-dichloro-2-propyl phosphate (TDCPP)) [17], endocrine disruption (TCEP and ACCEPTED MANUSCRIPT 4 TDCPP) [7, 18] and toxicity towards specific organs (TCEP for the kidney [7]). TDCPP has also been classified as carcinogenic, TCPP as carcinogenic to animals and TCEP as potentially carcinogenic [2, 19]. TCEP and TDCPP are subject to regulations in North America, Europe and Japan [20-22], and the European Chemicals Agency (ECHA) is currently preparing a proposal of restriction of use of these three chlorinated OPFRs [23]. Assessment of human exposure to OPFRs is usually performed through the determination of the precursor compounds and their metabolites in urine. In the case of chlorinated OPFRs, the main metabolites analysed in this matrix are the diesters bis(2chloroethyl) phosphate (BCEP) for TCEP; bis(chloropropyl) phosphate (BCPP) and bis(chloropropyl) hydroxypropyl phosphate for TCPP; and bis(1,3-dichloro-2-propyl) phosphate (BDCPP) for TDCPP [7, 16]. Although the analysis of urine is the most widely applied strategy in human biomonitoring, it is not exempt from limitations. It is subjected to ethical implications, restricted to a limited number of samples (what implies a limited population coverage) and affected by selection bias. Alternatively, the analysis of wastewater, understood as a pooled sample of urine of a whole community, provides chemical information that may help to understand exposure at the population level. Known as wastewater-based epidemiology (WBE), this methodology was initially implemented to gather information on the consumption of illicit drugs [24], and further extended to estimate human exposure to pesticides [25, 26], phthalates [27] and, very recently, OPFRs [28] through the determination of their human metabolites. An advantage inherent to WBE is that it is a non-intrusive methodology where one sample of wastewater represents thousands of urine samples, thus being inexpensive and more representative of the entire population. The promising results got in large-scale studies [29-31] highlights the need of developing analytical methods than can be applied ACCEPTED MANUSCRIPT 5 worldwide to establish WBE as a complementary tool for the monitoring of chemical exposure. Although there are several publications dealing with the determination of chlorinated OPFRs in wastewater [1, 6, 8], such application for their metabolites has been performed only in two occasions [28, 30]. Thereby, this study is aimed at optimizing, validating and applying a new analytical method to determine three biomarkers of exposure to chlorinated OPFRs (BCEP, BCPP and BDCPP) in wastewater. Conversely to the extended use of liquid chromatography (LC) - tandem mass spectrometry (MS/MS) for the separation and detection of OPFRs and their metabolites in urine [3235] and, very recently, wastewater [28], we suggest an alternative gas chromatography (GC) - high resolution mass spectrometry (HRMS) method after derivatization of the analytes to silyl-derivatives. GC-HRMS can afford further selectivity and qualitative information, improving the sensitivity in some cases. Therefore, the final objective is to present a GC-compatible solid-phase extraction (SPE) combined with a GC-based separation that can be applied to quantitatively determine BCEP, BCPP and BDCPP in wastewater. To the best of our knowledge, this is the first time that BCEP is included in a quantitative validated method for the determination of chlorinated OPFR metabolites in wastewater. 2. MATERIAL AND METHODS 2.1. Chemicals and reagents The structures of the chlorinated organophosphate diesters studied in this work and their precursor triesters are shown in Table S1. Individual standards of BCEP, BCPP (technical mixture of four isomers), BDCPP and their deuterated analogs (BCEP-d8, BCPP-d12 and BDCPP-d10) were supplied by Biozol (Munich, Germany). TCEP, ACCEPTED MANUSCRIPT 6 TCPP (also as technical mixture of four isomers) and TDCPP were supplied by SigmaAldrich (San Louis, Mi, USA). Their deuterated analogs (TCEP-d12, TCPP-d18 and TDCPP-d15) were supplied by Wellington Laboratories (Southgate Dr., Ontario, Canada). Mixed stock solutions containing the three diesters, the three diester deuterated analogs (used as surrogate or internal standards, IS), the three triesters or the three triester deuterated analogs were prepared in methanol (MeOH) and in ethyl acetate (EtOAc) and stored in the dark at -20 °C until use. HPLC-grade MeOH, acetic acid (100%) and ammonia (NH3) solution in ultrapure water (25%) were supplied by Merck (Darmstadt, Germany). EtOAc, formic acid (95-97%) and NH3 solution in MeOH (7 N) were supplied by Sigma-Aldrich (San Luis, Mi, USA). Ultrapure water was obtained in the laboratory by purifying demineralized water in a Milli-Q Gradient A-10 system (Merck-Millipore, Bedford, MA, USA). Silylation reagents, N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and N-tertbutyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) were provided by SigmaAldrich (San Louis, Mi, USA). 2.2. Sampling and sample treatment Composite raw wastewater samples of 24 h were collected at the inlet of an urban wastewater treatment plant (WWTP) that receives mostly domestic wastewater and serves a population of ~136,500 inhabitants in Santiago de Compostela (NW of Spain). Samples were collected in December 2017 and in March 2018 by a Sigma SD900 portable sampler from Hach (Loveland, CO, USA) working in time proportional mode. An aliquot of 120 mL was collected every 10 min from 9.00 a.m. to 9.00 a.m. of the following day. Composite samples were transferred to the laboratory and extracted within 8 h after the end of the sampling. ACCEPTED MANUSCRIPT 7 Aliquots (100 mL) were vacuum-filtered through 0.7 µm glass microfiber filters GF/A (Whatman, Kent, UK) and 0.45 µm cellulose filters (Merck-Millipore, Bedford, MA, USA) and spiked with 20 ng of BCEP-d8, BCPP-d12 and BDCPP-d10. An SPE procedure was developed for the extraction of the organophosphate diesters using the mixed-mode reversed-phase weak anion exchange sorbents Oasis WAX-150 mg (Waters, Milford, MA, USA). Sorbents were subsequently preconditioned with 6 mL of MeOH, 6 mL of ultrapure water and 6 mL of 2% of formic acid in ultrapure water to assure the protonation of the amine groups included in their polymeric structure. Samples were loaded at their natural pH and, after loading, sorbents were washed with 6 mL of ultrapure water and dried under nitrogen for ca. 30 min. A fractionated elution was performed: first, 4 mL of EtOAc were passed through the cartridges to remove interfering chemicals, including the precursor triesters. Then, analytes were recovered with 2 mL of EtOAc:MeOH:NH3 (83:15:2). Eluates were evaporated to dryness under nitrogen (99.999%), redissolved in 100 µL of EtOAc and filtered through 0.2 µm syringe-driven Nylon filters (Chmlab Group, Barcelona, Spain). An aliquot of 75 µL of each filtered extract was transferred into a glass micro-insert and mixed with 25 µL of MTBSTFA before injection. Samples were processed in triplicate. 2.3. Gas chromatography-high resolution mass spectrometry Instrumental analyses were performed using a GC-QTOF-MS system comprised of a 7890A gas chromatograph (Wilmington, DE, USA), a 7638B automatic sampler and a 7200 Quadrupole Time-of-Flight (Q-TOF) mass spectrometer from Agilent (Wilmington, DE, USA). Large-volume injections of 10 µL (2×5 µL) were made in solvent vent mode using a 10 µL-syringe and a Programmable Temperature Vaporizer (PTV) injector equipped with an Agilent ultra-inert liner containing glass wool. The ACCEPTED MANUSCRIPT 8 inlet temperature was increased from 60 ℃ (held for 0.6 min) to 300 ℃ (15 min) at a rate of 700 ℃/min. The flow rate through the split vent was set at 60 mL/min up to 0.6 min. After this time, the split valve was closed for 2 min and opened again with a purge flow of 100 mL/min. Chromatographic separation was carried out on a HP-5MS type capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness) supplied by Agilent Technologies. Helium (99.9999 %, Praxair, Spain) was used as carrier gas at a constant flow rate of 1.2 mL/min. The oven temperature programme was as follows: 60 ℃ (held for 2 min) ramped at 15 ℃/min to 280 ℃ (held for 5 min). The total run time was 21.67 min and the solvent delay 6 min. The transfer line, quadrupole and electron impact source were set at 280 ℃, 150 ℃ and 230 ℃, respectively. The mass spectrometer was operated in Electron Ionization (EI) mode at 70 eV and in single MS mode. The TOF analyzer worked in 2-GHz extended dynamic range mode, providing a resolution between 5500 at m/z 130.9915 and 9000 at m/z 413.9770. The mass axis was automatically recalibrated every 5 injections by infusion of a commercial solution of perfluorotributylamine in the EI source. Full scan high resolution mass spectra were recorded in centroid mode in the range from 50 to 500 m/z, at a frequency of 3.33 spectra/s. Table 1 displays retention times, empirical formulae and accurate m/z of the quantifier ions (Q) and qualifier ions (q) of the tert-butyl dimethylsilyl (TBDMS) derivatives of BCEP, BCPP, BDCPP and their deuterated analogs and of TCEP, TCPP, TDPP and their deuterated IS. Extracted ion chromatograms (EIC) of Q and q were reconstructed with a symmetric m/z window of 20 ppm. For BCPP and BCPP-d10, three peaks, corresponding to the three main isomers occurring in the commercial mixture, were obtained. The EI high-resolution mass spectra of the TBDMS forms of BCEP, BCPP, ACCEPTED MANUSCRIPT 15 EtOAc:MeOH:NH3 (83:15:2), therefore selected as elution solvent. Under these conditions, no insoluble residue was observed when extracting real wastewater. A matter of concern is the putative interference of precursor triesters, usually present in wastewater at high concentration levels. By comparing the responses of a standard of triesters before and after being mixed with 25% (v/v) of MTBSTFA, we verified that ca. 12% of TCEP and 8% of TCPP were degraded with the silylation reagent, giving to the formation of 0.7% and 1.7%, in molar basis, of the corresponding diesters. Conversely, TDCP turned out to be stable. To ensure the complete removal of triesters before the elution of diesters, an SPE of 100 mL of ultrapure water spiked with 100 ng TCEP, TCPP and TDCPP and 20 ng of their deuterated analogs (n=3) was performed following the protocol optimized for the extraction of diesters. Aliquots of 4 mL of EtOAc and 2 mL of EtOAc:MeOH:NH3 (83:15:2) were consecutively passed through the cartridges, collected separately, evaporated to dryness and reconstituted in 100 µL of EtOAc for instrumental analyses. 100% of the eluted triesters were recovered in the EtOAc fraction, rendered as an effective clean-up step for the determination of diesters in the basified EtOAc fraction. Additionally, a washing step of the sorbent with 6 mL of ultrapure water before drying the cartridges was kept in the final procedure to remove salts and inorganic interferences. 3.3. Method performance Method performance parameters are displayed in Table 2. The representation of the ratio analyte area/IS area versus analyte concentration fitted a linear model in the range IQL-1000 ng/mL with determination coefficients (R2) above 0.99. The analysis of instrumental blanks proved the absence of carryover or blank problems in the GC system. IDLs were between 0.38 ng/mL and 1.1 ng/mL, and IQLs between 1.3 ng/mL ACCEPTED MANUSCRIPT 16 and 3.6 ng/mL. Instrumental precision (both intraand inter-day) at 10 and 100 ng/mL was satisfactory for the three analytes, with %RSD values for six injections of a standard between 0.75% and 8.5%. MDLs, estimated as 3 times the SD of the procedural blanks for BCEP and BCPP and as the concentration in a sample providing a S/N of 3 for BDCPP, ranged from 1.1 to 4.6 ng/L. MQLs ranged from 4 to 15 ng/L, higher than the MQL reported for BDCPP (0.8 ng/L) by Been et al [30] but better than the MQL reached in their study for BCPP (15.4 ng/L), whereas BCEP could not be measured in that method. Trueness and precision of the whole SPE-GC-HRMS method, assessed through recovery experiments in ultrapure water and wastewater, were satisfactory for all the compounds: recoveries varied from 90% to 110% in ultrapure water spiked at 25 ng/L and from 90% to 100% in wastewater spiked at 500 ng/L. RSD varied between 3% and 13% in ultrapure water and between 1% and 6% in wastewater. Both recovery and %RSD values were acceptable according to the Commission Decision 2002/657/EC [44]. 3.4. Stability of diesters and triesters in wastewater Stability tests in real wastewater were conducted to assess: (i) the potential formation of BCEP, BCPP and BDCPP from their precursor organophosphate triesters occurring in wastewater; and (ii) the potential decrease in the concentration of BCEP, BCPP and BDCPP due to biodegradation and/or adsorption phenomena. Experiments were performed using unfiltered raw wastewater samples spiked only with triesters (objective (i)) or only with diesters (objective (ii)) and kept at room temperature for 48 h, a period of time longer than in-sewer residence and sampling. Figure 2 displays the responses of the investigated compounds relative to their average responses at time zero. For the ACCEPTED MANUSCRIPT 17 three triesters, the differences between the signals at different times were not statistically significant at the 95% of confidence level, proving that these compounds are stable. This observation is in agreement with previous findings reporting the nonelimination of chlorinated OPFRs during wastewater treatments, in contrast to the partial removal of some non-chlorinated OPFRs [45]. Moreover, the diesters were absent in the basic eluates from the triester stability study, proving that they are not formed from their precursor triesters in wastewater. In the diesters stability test, only the signal of BCEP underwent a significant increase at 6 h, followed by a slight decrease at 24 h and a stabilization afterwards. Been et al. had previously assessed the stability of some OPFRs metabolites in wastewater, although BCEP was not included in their study [28, 30]. They found that BCPP and BDCPP showed a decrease of 20% in the first 30 min at room temperature, but then appeared to stabilize in partial agreement with the results observed here. Therefore, the levels of BCEP, BCPP and BDCPP in sewage can be attributed to human metabolism, and they are neither expected to decrease due to biodegradation or adsorption processes nor expected to increase due to microbial or spontaneous hydrolysis of their precursor triesters (usually occurring in sewage [1, 46-48]). 3.5. Analysis of real samples Composite 24 h raw wastewater samples collected on two consecutive days in March 2018 were analyzed following the validated method. Only BCPP was found in both samples at levels of 63 ng/L and 64 ng/L, what implies population-normalized mass loads of 45 µg/day inhabitant and 47 µg/day inhabitant, respectively (daily flow rates: 97817 m3/day and 99917 m3/day; population served by the WWTP: 136,500 inhabitants). These values contrast with the study of Been et al. [30], in which they ACCEPTED MANUSCRIPT 18 assessed the levels of BCPP and BDCPP, among other analytes, in wastewaters from five European cities (Antwerp, Brussels, Geneva, Athens and Vilnius). They found an average concentration of BDCPP between 21 and 52 ng/L, with BCPP being <MQL (i.e. <15.4 ng/L) in all the analyzed samples. However, no Spanish cities were included in their study, a fact that could certainly contribute to the differences observed. It must be noticed that the levels reported in the current work correspond to two single days, and a larger wastewater sampling should be performed in future studies to properly establish an exposure estimation to organophosphate triesters following the WBE principles. Figure 3 shows the EIC of the analytes and the deuterated IS in a standard of 50 ng/mL and in a sample extract (IS level: 200 ng/mL). The use of HRMS allowed us to screen for the presence of other silylated phosphate esters through the search of the ion m/z 154.9924 (C2H8O4PSi). The EIC of this ion showed, for all samples, a high peak at 11.14 min that was further identified as dibutyl phosphate (DBP) by the acquisition and analysis of its analytical standard. Besides the characteristic base peak at m/z 154.9924, the EI spectrum shows other two ions at m/z 211.0556 (C6H16O4PSi) and m/z 267.1184 (C10H24O4PSi) corresponding to the monobutyl and dibutyl dimethyl silyl fragments (Figure S5). 4. CONCLUSIONS This study provides the first derivatization combined to GC-MS methodology developed for the determination of three chlorinated OPFR metabolites (BCEP, BCPP and BDCPP) in wastewater. Analyte separation and detection is conducted by GCHRMS after derivatization with MTBSTFA, a silylation reagent that provided an excellent derivatization efficiency even in complex wastewater extracts. The determination by HRMS with a QTOF system demonstrated a good performance in ACCEPTED MANUSCRIPT 19 terms of sensitivity, repeatability and reproducibility, further allowing to screen for other acidic organophosphate esters through the search of the ion m/z 154.9924, characteristic of the EI spectra of their silylated forms. The analysis of composite raw wastewater samples of 24 h collected in the NW of Spain showed, for the first time in this matrix, the presence of BCPP at levels over 60 ng/L. Appendix A. Supplementary material Declarations of interest: none Acknowledgements This work was financially supported by the Spanish Agencia Estatal de Investigación (project no. CTM2017-84763-C3-2-R), the Galician Council of Culture, Education and Universities (ED431C2017/36, VC predoctoral contract, ED481A-2017/156, and IGM postdoctoral contract, Plan Galego I2C-Modalidade B, ED481D 2017/003), Gil Dávila Foundation (VC research grant) and FEDER/ERDF. ACCEPTED MANUSCRIPT 20 References 1. T. Reemtsma, J.B. Quintana, R. Rodil, M. García-López, I. Rodríguez, Organophosphorus flame retardants and plasticizers in water and air I. Occurrence and fate, Trends Anal. Chem. 27(9) (2008) 727-737. http://dx.doi.org/10.1016/j.trac.2008.07.002. 2. I. van der Veen, J. de Boer, Phosphorus flame retardants: Properties, production, environmental occurrence, toxicity and analysis, Chemosphere 88(10) 2012 11191153. http://dx.doi.org/10.1016/j.chemosphere.2012.03.067. 3. M. Wu, G. Yu, Z. Cao, D. Wu, K. Liu, S. Deng, J. Huang, B. Wang, Y. Wang, Characterization and human exposure assessment of organophosphate flame retardants in indoor dust from several microenvironments of Beijing, China, Chemosphere 150 (2016) 465-471. http://dx.doi.org/10.1016/j.chemosphere.2015.12.111. 4. L. Zhou, M. Hiltscher, W. Püttmann, Occurrence and human exposure assessment of organophosphate flame retardants in indoor dust from various microenvironments of the Rhine/Main region, Germany, Indoor Air 27(6) (2017) 1113-1127. http://dx.doi.org/10.1111/ina.12397. 5. C. He, X. Wang, P. Thai, C. Baduel, C. Gallen, A. Banks, P. Bainton, K. English, J.F. Mueller, Organophosphate and brominated flame retardants in Australian indoor environments: Levels, sources, and preliminary assessment of human exposure. Environ. Pollut. 235 (2018) 670-679. http://dx.doi.org/10.1016/j.envpol.2017.12.017. 6. N. Ali, K. Shahzad, M.I. Rashid, H. Shen, I.M.I. Ismail, S.A.M.A.S. Eqani, Currently used organophosphate and brominated flame retardants in the environment of ACCEPTED MANUSCRIPT 21 China and other developing countries (2000–2016), Environ. Sci. Pollut. Res. 24(23) (2017) 18721-18741. http://dx.doi.org/10.1007/s11356-017-9336-3. 7. A.M. Saillenfait, S. Ndaw, A. Robert, J.P. Sabaté, Recent biomonitoring reports on phosphate ester flame retardants: a short review, Arch. Toxicol. 92(9) (2018) 2749-2778. http://dx.doi.org/10.1007/s00204-018-2275-z. 8. G.L. Wei, D.Q. Li, M.N. Zhuo, Y.S. Liao, Z.Y. Xie, T.L. Guo, J.J. Li, S.Y. Zhang, Z.Q. Liang, Organophosphorus flame retardants and plasticizers: Sources, occurrence, toxicity and human exposure, Environ. Pollut. 196 (2015) 29-46. http://dx.doi.org/10.1016/j.envpol.2014.09.012. 9. M. Abou-Elwafa Abdallah, G. Pawar, S. Harrad, Human dermal absorption of chlorinated organophosphate flame retardants; implications for human exposure, Toxicol. Appl. Pharmacol. 291 (2016) 28-37. http://dx.doi.org/10.1016/j.taap.2015.12.004. 10. A. Bello, C.C. Carignan, Y. Xue, H.M. Stapleton, D. Bello, Exposure to organophosphate flame retardants in spray polyurethane foam applicators: Role of dermal exposure, Environ. Int. 113 (2018) 55-65. http://dx.doi.org/10.1016/j.envint.2018.01.020. 11. M. Frederiksen, H.M. Stapleton, K. Vorkamp, T.F. Webster, N.M. Jensen, J.A. Sørensen, P.A. Clausen, J.B. Nielsen, Dermal uptake and percutaneous penetration of organophosphate esters in a human skin ex vivo model, Chemosphere 197 (2018) 185-192. http://dx.doi.org/10.1016/j.chemosphere.2018.01.032. 12. E. Mendelsohn, A. Hagopian, K. Hoffman, C.M. Butt, A. Lorenzo, J. Congleton, T.F. Webster, H.M. Stapleton, Nail polish as a source of exposure to triphenyl ACCEPTED MANUSCRIPT 22 phosphate, Environ. Int. 86 (2016) 45-51. http://dx.doi.org/10.1016/j.envint.2015.10.005. 13. J.B. Quintana, M. Rosende, R. Montes, T. Rodríguez-Álvarez, R. Rodil, R. Cela, M. Miró, In-vitro estimation of bioaccessibility of chlorinated organophosphate flame retardants in indoor dust by fasting and fed physiologically relevant extraction tests, Sci. Total Environ. 580 (2017) 540-549. http://dx.doi.org/10.1016/j.scitotenv.2016.11.210. 14. E.D. Schreder, N. Uding, M.J. La Guardia, Inhalation a significant exposure route for chlorinated organophosphate flame retardants, Chemosphere 150 (2016) 499504. http://dx.doi.org/10.1016/j.chemosphere.2015.11.084. 15. F. Xu, G. Giovanoulis, S. van Waes, J.A. Padilla-Sanchez, E. Papadopoulou, J. Magnér, L.S. Haug, H. Neel, A. Covaci, Comprehensive Study of Human External Exposure to Organophosphate Flame Retardants via Air, Dust, and Hand Wipes: The Importance of Sampling and Assessment Strategy, Enviro. Sci. Technol. 50(14) (2016) 7752-7760. http://dx.doi.org/10.1021/acs.est.6b00246. 16. R. Hou, Y. Xu, Z. Wang, Review of OPFRs in animals and humans: Absorption, bioaccumulation, metabolism, and internal exposure research, Chemosphere 153 (2016) 78-90. http://dx.doi.org/10.1016/j.chemosphere.2016.03.003. 17. A. Araki, I. Saito, A. Kanazawa, K. Morimoto, K. Nakayama, E. Shibata, M. Tanaka, T. Takigawa, T. Yoshimura, H. Chikara, Y. Saijo, R. Kishi, Phosphorus flame retardants in indoor dust and their relation to asthma and allergies of inhabitants, Indoor Air 24(1) (2014) 3-15. http://dx.doi.org/10.1111/ina.12054. 18. J.D. Meeker, H.M. Stapleton, House Dust Concentrations of Organophosphate Flame Retardants in Relation to Hormone Levels and Semen Quality Parameters, ACCEPTED MANUSCRIPT 23 Environ. Health Perspect. 118(3) (2010) 318-323. http://dx.doi.org/10.1289/ehp.0901332. 19. World Health Organization, Flame retardants: Tris(Chloroproyl) Phosphate and Tris(2-Chloroethyl) Phosphate, International Programme on Chemical Safety, Geneva, Switzerland, 1998. 20. Environmental Protection Agency, Office of Environmental Health Hazard Assessment, Safe Drinking Water and Toxic Enforcement Act of 1986. Chemicals known to the State to cause cancer or reproductive toxicity, December 29, 2017, https://oehhacagov/media/downloads/proposition-65/p65122917pdf, 2017 (accessed on 15/11/2018). 21. Ministry of Justice, Canada Consumer Product Safety Act, Consolidation SC 2010, c. 21 Current to March 26, 2018 Last amended on December 12, 2016, https://laws-loisjusticegcca/PDF/C-168pdf, 2018 (accessed on 15/11/2018). 22. European Chemicals Agency, https://echa.europa.eu/, 2018 (accessed on 15/11/2018). 23. European Chemicals Agency, https://echa.europa.eu/registry-of-restrictionintentions/-/dislist/details/0b0236e1829a30b8, 2018 (accessed on 15/11/2018). 24. C. Ort, A.L.N. Nuijs, J.D. Berset, L. Bijlsma, S. Castiglioni, A. Covaci, P. Voogt, E. Emke, D. Fatta-Kassinos, P. Griffiths, F. Hernández, I. González-Mariño, R. Grabic, B. Kasprzyk-Hordern, N. Mastroianni, A. Meierjohann, T. Nefau, M. Östman, Y. Picó, I. Racamonde, M. Reid, J. Slobodnik, S. Terzic, N. Thomaidis, K.V. Thomas, Spatial differences and temporal changes in illicit drug use in Europe quantified by wastewater analysis, Addiction 109(8) (2014) 1338-13352. http://dx.doi.org/10.1111/add.12570. ACCEPTED MANUSCRIPT 24 25. N.I. Rousis, E. Zuccato, S. Castiglioni, Monitoring population exposure to pesticides based on liquid chromatography-tandem mass spectrometry measurement of their urinary metabolites in urban wastewater: A novel biomonitoring approach, Sci. Total Environ. 571 (2016) 1349-1357. http://dx.doi.org/10.1016/j.scitotenv.2016.07.036. 26. N.I. Rousis, E. Zuccato, S. Castiglioni, Wastewater-based epidemiology to assess human exposure to pyrethroid pesticides, Environ. Int. 99 (2017) 213-220. http://dx.doi.org/10.1016/j.envint.2016.11.020. 27. I. González-Mariño, R. Rodil, I. Barrio, R. Cela, J.B. Quintana, Wastewater-Based Epidemiology as a New Tool for Estimating Population Exposure to Phthalate Plasticizers, Environ. Sci. Technol. 51(7) (2017) 3902-3910. http://dx.doi.org/10.1021/acs.est.6b05612. 28. F. Been, M. Bastiaensen, F.Y. Lai, A.L.N. van Nuijs, A. Covaci, Liquid Chromatography–Tandem Mass Spectrometry Analysis of Biomarkers of Exposure to Phosphorus Flame Retardants in Wastewater to Monitor CommunityWide Exposure, Anal. Chem. 89(18) (2017) 10045-10053. http://dx.doi.org/10.1021/acs.analchem.7b02705. 29. N.I. Rousis, E. Gracia-Lor, E. Zuccato, R. Bade, J.A. Baz-Lomba, E. Castrignanò, A. Causanilles, A. Covaci, P. de Voogt, F. Hernández, B. Kasprzyk -Hordern, J. Kinyua, A.K. McCall, B.G. Plósz, P. Ramin, Y. Ryu, K.V. Thomas, A. van Nuijs, Z. Yang, S. Castiglioni, Wastewater-based epidemiology to assess pan-European pesticide exposure, Water Res. 121 (2017) 270-279. http://dx.doi.org/10.1016/j.watres.2017.05.044. 30. F. Been, M. Bastiaensen, F.Y. Lai, K. Libousi, N.S. Thomaidis, L. Benaglia, P. Esseiva, O. Delémont, A.L.N. van Nuijs, A. Covaci, Mining the Chemical ACCEPTED MANUSCRIPT 31 Table 1. Retention times (RT), empirical formulae, accurate m/z values of the ions selected as quantifier (Q) and qualifier (q) and ratio between both ions (%). Analyte RT Quantifier ion (Q) Qualifier ion (q) q/Q ratio (%) Formula m/z Formula m/z BCEP-TBDMS 11.67 C2H8O4PSi 154.9924 C4H11ClO4PSi 216.9847 47 BCPP-TBDMS 11.88/11.97/12.04a C2H8O4PSi 154.9924 C5H13ClO4PSi 231.0004 11b BDCPP-TBDMS 14.09 C2H8O4PSi 154.9924 C5H12Cl2O4PSi 264.9614 19 BCEP-d8-TBDMS 11.63 C2H6D2O4PSi 157.0050 ꟷ ꟷ ꟷ BCPP-d12 -TBDMS 11.82/11.90/11.97a C2H6D2O4PSi 157.0050 ꟷ ꟷ ꟷ BDCPP-d10-TBDMS 14.03 C2H6D2O4PSi 157.0050 ꟷ ꟷ ꟷ TCEP 11.50 H4O4P 98.9842 C2H5ClO3P 142.9659 116 TCPP 11.76/11.86/11.94a H4O4P 98.9842 C2H6O4P 124.9998 105b TDCPP 14.02 H4O4P 98.9842 C3H6Cl2O3P 190.9426 51 TCEP-d12 11.45 D4O4P 103.0093 ꟷ ꟷ ꟷ TCPP-d18 11.70/11.80/11.88a D4O4P 103.0093 ꟷ ꟷ ꟷ TDCPP-d15 14.96 D4O4P 103.0093 ꟷ ꟷ ꟷ a RT of the three main isomers b q/Q ratio calculated considering the three main isomers ACCEPTED MANUSCRIPT 32 Table 2. Method performance parameters: linearity, intraand inter-day instrumental precision, instrumental quantification and detection limits (IQL and IDL), trueness, method precision and method detection and quantification limits (MDL and MQL). Analyte Linearity Intra-day precision (%RSD)b Inter-day precision (%RSD)b IDL IQL (R2)a 10 ng/mL 100 ng/mL 10 ng/mL 100 ng/mL (ng/mL) (ng/mL) BCEP 0.9988 6.6 2.6 8 8.5 1.1 3.6 BCPP 0.9988 4.1 0.75 4.1 5.3 0.38 1.3 BDCPP 0.9997 3.4 1.5 2.8 2.5 0.81 2.7 Analyte Trueness and precision (%R and %RSD)c MDL MQL Ultrapure water (25 ng/L) Wastewater (500 ng/L) (ng/L) (ng/L) BCEP 110 (13) 100 (1) 1.1 4 BCPP 96 (3) 90 (2) 1.2 4 BDCPP 90 (5) 98 (6) 4.6 15 a Determination coefficient for a 10-point calibration curve. Linear range: IQL - 1000 ng/mL b Relative standard deviation (%) for six injections of a standard over 24 h (intra-day precision) or four weeks (inter-day precision) c Average recovery (%R) from the nominal spiking value and %RSD from the average measured concentration; experiments performed in triplicate ACCEPTED MANUSCRIPT Supplementary Material to: Determination of human metabolites of chlorinated phosphorous flame retardants in wastewater by N-tert-butyldimethylsilyl-Nmethyltrifluoroacetamide-derivatization and gas chromatography-high resolution mass spectrometry Verónica Castro, Rosario Rodil, José Benito Quintana*, Rafael Cela, Laura SánchezFernández, Iria González-Mariño* Department of Analytical Chemistry, Nutrition and Food Sciences, IIAA – Institute for Food Analysis and Research, Universidade de Santiago de Compostela, Constantino Candeira S/N, 15782 – Santiago de Compostela, Spain. *Corresponding author: Iria González Mariño phone: +34 881 816035 E-mail: [email protected] *Co-corresponding author: José Benito Quintana phone: +34 881 816035 E-mail: [email protected] Table S1. Organophosphate diesters considered in this work and their corresponding parent triesters. N.B.: BCPP and TCPP comprise a mixture of isomers, the most abundant one only being shown. Diester Structure Triester Structure Bis(2-chloroethyl) phosphate (BCEP) Tris(2-chloroethyl) phosphate (TCEP) Bis(1-chloro-2-propyl) phosphate (BCPP) Tris(1-chloro-2-propyl) phosphate (TCPP) Bis(1,3-dichloro-2-propyl) phosphate (BDCPP) Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) 4 x10 0 1 2 3 4 157.0048 287.0270 222.0163 73.0465 173.9646 244.1550 130.0678 88.0565 321.1831 371.1800 115.0905 261.0765 4 x10 0 1 2 3 4 5 6 7 157.0049 238.0446 73.0466 325.1575 207.0324 123.9724 261.1697 173.9634 298.1411 4 x10 0 0.5 1 1.5 2 2.5 3 3.5 157.0047 270.9993 79.0225 299.2399 386.9912 234.0161 173.9645 136.9817 207.0323 92.9920 62.0634 327.3037 Counts vs. Mass-to-Charge (m/z) 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 Figure S1. High-resolution mass spectra (EI) of the tert-butyl dimethylsilyl derivatives of BCEP-d8, BCPPd12 and BDCPP-d10. Figure S2. High-resolution mass spectra (EI) of the trimethylsilyl derivatives of BCEP, BCPP and BDCPP. BCEP-d8-TBDMS BCPP-d12-TBDMS BDCPP-d10-TBDMS 5 x10 0 1 2 3 4 5 154.9932 259.0320 216.9873 172.9619 77.0070 278.9773 136.9846 62.9998 5 x10 0 2 4 6 154.9929 197.0398 171.0240 77.0078 273.0475 231.0012 5 x10 0 0.5 1 1.5 154.9929 264.9686 75.0259 171.0235 342.9678 136.9857 Counts vs. Mass - to - Charge (m/z) 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 P HO OH O O Si P HO OH O O Si P HO OH O O Si P O HO O O Cl Si P O O O O Cl Si Cl P O OH O O Si Cl P O HO O O Cl Cl Si P O O O O Cl Cl Si Cl Cl BCEP-TMS BCPP-TMS BDCPP-TMS Figure S3. Comparison of the extracted ion chromatogram (EIC) of the three organophosphate diesters in a 100 ng/mL standard derivatized with 25% of MSTFA (green) and with 25% of MTBSTFA (blue). Acquired after a GC separation with the following temperature programme: initially 60 ℃, ramped at 10 ℃/min to 280 ℃ (held for 5 min); injection in splitless mode. Figure S4. Height of individual peaks for the organophosphate diesters-TBDMS derivatives when injecting 2 µL in splitless mode versus 10 µL in large-volume injection mode. BCPP 1 to 3 represent the area of the three main isomers of BCPP. 0 2000000 4000000 6000000 8000000 10000000 12000000 14000000 BCEP BCPP 1 BCPP 2 BCPP 3 BDCPP Height 2 µL 10 µL MSTFA MTBSTFA BCPP-TMS BCEP-TBDMS BCPP-TBDMS BDCPP-TBDMS BCEP-TMS BDCPP-TMS Figure S5. Extracted ion chromatogram (EIC) of m/z 154.9924 and EI MS spectra of the peak at 11.14 min in a standard of dibutyl phosphate-TBDMS (upper chromatogram and spectrum) and in a real sample (lower chromatogram and spectrum). 6 x10 0 2 4 6 +EI EIC(154.9924) Scan 11.145 6 x10 0 1 2 3 +EI EIC(154.9924) Scan 11.154 Counts vs. Acquisition Time (min) 10,85 10,9 10,95 11 11.05 11.1 11.15 11.2 11,25 11.3 11.35 11,4 11,45 6 x10 0 0.5 1 1.5 154.9929 77.0067 211.0560 267.1175 Counts vs. Mass - to - Charge (m/z) 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 18 8 .1470 6 x10 0 0.1 0.5 1 1.5 154.9930 211.0553 77.0071 267.1178 Counts vs. Mass - to - Charge (m/z) 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 241.1617 144.0838 P HO OH O O Si Standard Standard Sample Sample P HO OH O O Si