Investigation of in vitro biotransformation of tris (1-chloro-2-propyl) phosphate and confirmation in human urine

SI-1 SUPPLEMENTARY INFORMATION Investigation of in vitro biotransformation of tris (1-chloro-2propyl) phosphate and confirmation in human urine Fatima den Ouden1, Andrea Estévez-Danta2, Lidia Belova1, Celine Gys1, Anna Klimowska1,3, Maarten Roggeman1, Natan Van Wichelen1, José Benito Quintana2, Rosario Rodil2, Giulia Poma1, Adrian Covaci1 1 Toxicological Centre, University of Antwerp, 2610, Wilrijk 2Department of Analytical Chemistry, Nutrition and Food Chemistry. Institute of Research on Chemical and Biological Analysis (IAQBUS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain 3Department of Toxicology, Faculty of Pharmacy, Medical University of Gdańsk, Gdańsk, Poland *Corresponding authors E-mail addresses: [email protected] (F. den Ouden) and [email protected] (A. Covaci) SI-2 SI-1 Chemicals and reagents Pooled HLMs (n=200, mixed gender, 20 mg/mL) and human liver cytosol (HLCYT) (n=50, mixed gender, 10 mg/mL) were obtained from Xenotech (Kansas City, USA). TCEP-d12 was custom synthesized by dr. Vladimir Belov (Max Planck Institute, Göttingen, Germany). DPHP-d10, 2,5uridinediphosphate glucuronic acid (UDPGA), adenosine-3′-phosphate 5′- phosphosulfate (PAPS, > 60 %) lithium salt hydrate, alamethicin (neat, > 98 %), dimethyl sulfoxide (DMSO), phenacetin and 4nitrophenol (4-NP) were acquired from Sigma-Aldrich (Bornem, Belgium). TCIPP (analytical standard, purity > 99%) was obtained from LGC, Dr Ehrenstorfer (Augsburg, Germany). Acetonitrile (ACN, HPLC-grade) and methanol (MeOH, LC-MS grade, ≥99.9%) were purchased from Biosolve (Valkenswaard, the Netherlands). Nicotinamide-adenine-dinucleotide phosphate (NADPH) salt (97%), formic acid (>98%) and hydrochloric acid (37%) were obtained from Merck KGaA (Darmstadt, Germany). β-glucuronidase (lyophilized powder from Escherichia coli, >10 000 000 unit/g) were obtained from Sigma-Aldrich (Bornem, Belgium). Ultrapure water was obtained from a PURELAB Flex system (ρ=18.2 MΩ/cm, Elga Veolia, Tienen, Belgium). Trizma buffer (100 mM) was prepared by dissolving 12.2 g Trizma base and 1.02 g MgCl2 6 H2O in 1 L ultrapure water. The pH was adjusted to 7.4 by addition of 1 M HCl solution. SI-3 SI-2 Tables Table S1, concentrations (ng/mL) in each sample determined by semi-quantitative analysis on the LC-QTOF-MS. Concentration (ng/mL) BCIPHIPP BCIPP TCIPP-M1 TCIPP-M3 1 65.7 <0.5 3.5 3.6 2 289.6 <0.5 8.9 13.1 3 57.8 <0.5 2.5 5.5 4 92.3 <0.5 2.6 3.2 5 591.4 <0.5 14.7 26.4 6 73.5 <0.5 1.3 2.8 7 173.3 <0.5 5.1 8.0 8 155.5 <0.5 5.8 5.2 9 85.7 <0.5 <0.05 2.3 10 104.3 1.53 1.6 5.5 11 Detected* <0.5 <0.05 Detected* 12 346.7 0.84 5.8 13.9 13 12.2 <0.5 <0.05 0.1 14 10.6 <0.5 0.1 <0.05 *In this sample BCIPHIPP and TCIPP-M3 were detected, but due to a low score for the internal standard it was not possible to use the area of the IS to calculate concentrations. SI-4 SI-3 Figures Figure SI-1 Extracted ion chromatogram [M+H]+ and molecular structure (top) and MS/MS spectrum (CE 10V) (bottom) of phenacetin. Found m/z values and mass errors of fragments are displayed. The obtained MS/MS spectrum was compared with experimental data from libraries leading to L2 according to the confidence levels proposed by Schymanski et al., 2014. SI-5 Figure SI-2 Extracted ion chromatogram [M+H]+ and molecular structure (top) and MS/MS spectrum (CE 10 eV) (bottom) of phenacetin-M1. Found m/z values and mass errors of fragments are displayed. The obtained MS/MS spectrum was compared with experimental data from libraries leading to L2 confirmation. SI-6 Figure SI-3 Extracted ion chromatogram [M+H]+ and molecular structure (top) and MS/MS spectrum (CE 10 eV) (bottom of phenacetin-M2. Found m/z values and mass errors are indicated. The obtained MS/MS spectrum was compared with in silico predicted spectra leading to L3 confirmation. SI-7 Figure SI-4 Extracted ion chromatogram [M-H]- and molecular structure (top) and MS/MS spectrum (CE 10 eV) (bottom) of 4-nitrophenol in the glucuronidation samples. Found m/z values and mass errors of fragments are displayed. The obtained MS/MS spectrum was compared with experimental data from libraries leading to L2 confirmation. SI-8 Figure SI-5 Extracted ion chromatogram [M-H]- and molecular structure (top) and MS/MS spectrum (CE 10 eV) (bottom) of 4-nitrophenol-glucuronide in the glucuronidation samples. Found m/z values and mass errors are indicated. The obtained MS/MS spectrum was compared with in silico predicted spectra leading to L3 confirmation. SI-9 Figure SI-6 Extracted ion chromatogram [M-H]- and molecular structure (top) and MS/MS spectrum (CE 10 eV) (bottom) of 4-nitrophenol in the sulfation samples. Found m/z values and mass errors of fragments are displayed. The obtained MS/MS spectrum was compared with experimental data from libraries leading to L2 confirmation. SI-16 Figure SI-13, Chromatogram [M+H]+ and molecular structure of bis (1-chloro-2-propyl) hydrogen phosphate (BCIPP) in standard (top) and urine sample (bottom) with the black arrow indicating the peak of BCIPP. No MS/MS spectra from BCIPP in urine samples could be obtained. Retention time was compared with injection of an analytical standard leading to L2c confirmation as proposed by Roggeman et al., 2022. SI-17 Figure SI-134 Chromatogram [M+H]+ and molecular structure of carboxyethyl bis (1-chloro-2-propyl) phosphate (TCIPPM1) in a urine sample. SI-18 Figure SI-145 Predicted MS/MS spectra for carboxyethyl bis (1-chloro-2-propyl) phosphate (TCIPP-M1) by CFM-ID (top) and MS/MS spectrum of TCIPP-M1 in urine samples (bottom) at 10 eV. Found m/z values, mass errors and proposed structures are indicated. Comparing obtained and predicted spectra led to L3 confirmation. SI-19 Figure SI-156 Chromatogram [M+H]+ and molecular structure of 1-chloro-3-hydroxypropan-2-yl bis (1-chloropropan-2-yl) phosphate (TCIPP-M3) in a urine sample. SI-20 Figure SI-17 Predicted MS/MS spectra for 1-chloro-3-hydroxypropan-2-yl bis (1-chloropropan-2-yl) phosphate (TCIPPM3) by CFM-ID (top) and obtained MS/MS spectra of TCIPP-M3 in a urine sample (bottom) at 10 eV. Found m/z values, mass errors and proposed structures are indicated. Comparing obtained and predicted spectra led to L3 confirmation. SI-21 Figure SI-18 Obtained calibration curve of 1-hydroxy-2-propyl bis (1-chloro-2-propyl) phosphate (BCIPHIPP) for semiquantification of tris (1-chloro-2-propyl) phosphate (TCIPP) metabolites. On the y-axis the ratio between the area of BCIPHIPP and the area of internal standard is displayed. On the x-axis the BCIPHIPP concentration can be seen. A linear calibration curve was constructed with the intercept forced through 0. The formula for the calibration curve was y=0.3853x (R2=0.9984) in which 0.3853 is the slope which was used as response factor for further calculations of carboxyethyl bis (1chloro-2-propyl) phosphate (TCIPP-M1) and 1-chloro-3-hydroxypropan-2-yl bis (1-chloropropan-2-yl) phosphate (TCIPPM3) concentrations. SI-3 References Roggeman, M., Belova, L., Fernández, S. F., Kim, D. H., Jeong, Y., Poma, G., Remy, S., Verheyen, V. J., Schoeters, G., van Nuijs, A. L. N., & Covaci, A. (2022). Comprehensive suspect screening for the identification of contaminants of emerging concern in urine of Flemish adolescents by liquid chromatography high-resolution mass spectrometry. Environmental Research, 214(March). https://doi.org/10.1016/j.envres.2022.114105 Schymanski, E. L., Jeon, J., Gulde, R., Fenner, K., Ru, M., Singer, H. P., & Hollender, J. (2014). Identifying Small Molecules via High Resolution Mass Spectrometry: Communicating Confidence. Environmental Science and Technology, 48, 2097–2098. https://doi.org/10.1021/es5002105 y = 0.3853x R² = 0.9984 0 10 20 30 40 50 60 70 80 90 050 100 150 200 250 Ratio Area BCIPHIPP/Area IS BCIPHIPP concentration (ng/mL) Calibration BCIPHIPP