2H-SIAM for 6PPD transformation product in soil
Description
This is the dataset for the manuscript named with "H/D Exchange Coupled with 2H-labeled Stable Isotope Assisted Metabolomics Discover Transformation Products of 6PPD in Soils". Quantitative data of HRMS were provided here. The abstract for the manuscript is as following: 6PPD ((N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine) and its transformation products (TPs), 6PPD-quinone (6PPDQ), have raised worldwide concern due to that 6PPDQ induced high mortality in coho salmon. In this study, TPs of 6PPD in soils were firstly explored by the coupling use of Hydrogen/Deuterium (H/D) exchange, High-Resolution Mass Spectrometry (HRMS), and 2H-labeled Stable Isotope Assisted Metabolomics (2H-SIAM). 6PPD-d9 was synthesized by H/D exchange, and 9 TPs of 6PPD were annotated with identification confidences level 2a or 2b by 2H-SIAM pipeline. Among them, 6PPDQ and PPPD (N-phenyl-p-phenylenediamine), were further identified by commercially available standards with confidences level 1. The toxicity of 6PPD, 6PPDQ and PPPD were further evaluated by the acute toxicity tests in adult zebrafish (Danio rerio), zebrafish embryo, and Vibrio fischeri. 6PPDQ did not exhibit acute toxicity to the tested species even at very high nominal concentration, and toxicity of 6PPD and PPPD were more ubiquitous, indicating their differences in toxic mechanism. Furthermore, possible new formation and toxic mechanism for 6PPDQ were proposed with preliminary data, and further investigation is necessary. In summary, this study highlighted the new strategy and its performance in discovering TPs of contaminants of emerging concern (CECs), and ecotoxicology study contributed to understanding the fate of 6PPD and its TPs in the environment.
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Steps to reproduce
6PPD and synthesized 6PPD-d9 were added to one-quarter of the soils, and they were further mixed with the rest soils to obtain soils with a final content of 50 mg/kg 6PPD or 6PPD-d9. Soils were incubated in Petri dishes in the lab and watered with Milli-Q water weekly to keep moisture. After 30 days of incubation at room temperature, soils were harvested and dried with a lyophilizer. Then, they were extracted by microwave extraction using acetone and hexane (1:3, v/v, 10.0 mL for 1.0 g soil). The soil extract was dried using anhydrous Na2SO4 and finally concentrated to 1.0 mL under nitrogen flow. Subsequently, extract from 6PPD and 6PPD-d9 treated soil were mixed with the ratio of 1:3 and 3:1 to obtain Mix1:3 and Mix3:1 samples. The mixed samples were then analyzed by UPLC-ESI-HRMS, Ultimate 3000 (Dionex) coupled with a Q Exactive Plus Orbitrap mass spectrometers (ThermoFisher Scientific, U.S.A) and heated electrospray ionization (ESI) source. Chromatographic condition was as following: 5 μL of samples was injected into the UPLC-ESI-HRMS system. UPLC solvents were A, water with 3.8 mM ammonium acetate, and B, methanol. UPLC were performed at 1 mL/min at 25 ℃ with the following linear gradient (minutes, %B): 0, 10%; 2, 10%; 5, 95%; 20, 95%; 21, 10%; 22, 10%. MS1 spectrum was detected via profile mode with a resolving power of 70,000 FWHM (full width at half maxima) at m/z 200 and an automatic gain control setting of 3 × 106 with a maximum injection time of 200 ms. The heated electrospray ionization (ESI) source was operated using the following settings: positive model, sheath gas flow rate, 40 au; auxiliary gas flow rate, 20 au; spray voltage, 3.8 kV; capillary temperature, 325 ℃. Mass spectrum raw data were firstly formatted to the file format ".mzXML" by ProteoWizard (3.0.20353x86_64) MSConvertGUI(64-bit) . The following models were sequentially used in MZmine2.53 data processing: mass detection, ADAP Chromatogram, deconvolution, isotopic peak grouper, RT calibration, align, duplicate peak filter, feature list rows filter, same RT and m/z range gap filler, and standard compound normalizer. Sort sequence alphabetically if necessary. Feature list information was exported as .csv format with m/z, RT and height information from MZmine2. After that, 2H-SIAM(1.0) was used to extract feature pairs of isotopologues as following: Mix1:3 and Mix3:1, it was imported into 2H-SIAM(1.0) with the following setting: Mass tolerance 20 ppm, RT tolerance 0.4 min, Number of labeled atom 3-9, Mass difference of atoms between labeled or not (Da) 1.006174, Ratio for F1, F2 and F3, 0.3333, 3 and 3, Tolerance for F1, F2 and F3, 0.15, 0.15 and 0.5. The annotated features were further confirmed by their MS2 spectrums. The MS2 spectrums were obtained by UPLC-ESI-HRMS in data dependent acquisition (DDA) model with a 0.4 m/z isolation window and nominal collision energy of 40 with the scanning resolution of 17500 FWHM at both MS1 and MS2 mass detector.