Manufacturer-Specific Isotopologue Fingerprints and Hydrolysis Kinetics of Imidacloprid: High-Resolution Orbitrap Mass Spectrometry Data and Statistical Analysis

This dataset contains high-resolution mass spectrometry data and associated analytical workflows for measuring the isotopologue ratios of imidacloprid (IMI) at its natural isotopic abundance. The data support manufacturer source identification through isotopic fingerprinting and determination of kinetic isotope effects during alkaline hydrolysis. Dataset Components 1. Manufacturer_Fingerprinting/ MS1/ - Full-scan MS¹ Analysis Full-scan MS¹ isotopologue analysis of intact imidacloprid from seven commercial manufacturers: LGC Chem Cruz Sigma Thermo HPC Target Mol Roth Contents: RAW/: Thermo RAW files acquired on Orbitrap Exploris 240 Resolution: 120,000 @ m/z 200 Microscans: 10 µscans AGC target: 1×10⁶ Mode: Positive electrospray ionization (ESI+) data/: 25 ISOX files (processed isotopologue data extracted via IsoX software) isotopologs.tsv: Target m/z definitions for monitored isotopologues: M0 (monoisotopic mass) ¹³C (single carbon-13 substitution) ¹⁵N (single nitrogen-15 substitution) ³⁷Cl (single chlorine-37 substitution) Combination isotopologues (¹³C¹⁵N, ³⁷Cl¹³C, ³⁷Cl¹⁵N) sequence.csv: Sample metadata with manufacturer labels and injection sequence Referenciation.csv: Reference material δ-values for international isotope scales (VPDB for carbon, Air for nitrogen, VSMOC for chlorine) flow_injection_w_syringe_M0-V4_AS_Calibration.Rmd: R Markdown workflow for data processing Bracketing-based delta value calculation with drift correction Full error propagation (standard error, shot noise estimates) Quality control metrics and visualization MS2/ - Fragment-specific MS² Analysis Fragment-specific MS² isotopologue analysis following collision-induced dissociation (CID) Fragment 84/, Fragment 175/, Fragment 209/Each folder contains data for a specific fragment ion: RAW files (Orbitrap Exploris 240, ddMS² mode) ISOX files (n=25 per fragment, one per sample/replicate) isotopologs.tsv with fragment-specific isotopologue definitions Sequence metadata files Fragments analyzed: m/z 84: Imidazolidine ring fragment m/z 175: Chloropyridine moiety m/z 209: Dechlorinated parent fragment R_Scripts_PCA/ Statistical analysis scripts for multivariate data analysis: 20251014_One_Way_Anova_Welch_Data_Revised_3.R ANOVA-based variable selection Identifies manufacturer-discriminating isotopologue ratios Welch's test implementation for unequal variances Multiple testing correction 20251014_PCA_Rework_Final_Rescale_4_Dataset_1.R 20251014_PCA_Rework_Final_Rescale_4_Dataset_2.R Principal component analysis with centered/scaled data transformation Eigenvalue decomposition and variance explained calculations Publication-ready visualizations: PCA scores plots with manufacturer labels Scree plots showing variance explained per PC Loading biplots for variable contributions Hierarchical clustering dendrograms 2. Imidacloprid_Hydrolysis/ Alkaline hydrolysis kinetics experiment for determination of compound-specific isotope analysis (CSIA) enrichment factors. Experimental conditions: pH 12 (NaOH solution) Temperature: 30°C Time-resolved sampling across hydrolysis time course Contents: combined.isox: Merged isotopologue data across all sampling time points isotopologs.tsv: Target m/z definitions for intact imidacloprid isotopologues (M0, ¹³C, ¹⁵N, ³⁷Cl) 20250623_IsoOrbi_Dua-Inlet_AutoPath_AutoEval_V7.R: Dual-inlet data processing script using the isoorbi R package Automated block definition (reference/sample alternation) Outlier flagging based on AGC thresholds Delta value calculation with bracketing standards Kinetic isotope fractionation factor determination (εC and εN) Instrument: Orbitrap Q Exactive with dual-inlet configuration Purpose: Determination of carbon (εC) and nitrogen (εN) isotope enrichment factors during alkaline hydrolysis Methodological Details Instrumentation Orbitrap Exploris 240 (Manufacturer fingerprinting experiments) Full MS and ddMS² acquisition modes Resolution: 120,000 @ m/z 200 Mass analyzer: Orbitrap Ionization: Positive electrospray ionization (ESI+) AGC target: 1×10⁶ Microscans: 10 Orbitrap Q Exactive (Hydrolysis experiments) Dual-inlet configuration for reference-sample bracketing High-resolution accurate mass (HRAM) detection Continuous monitoring mode Sample Introduction Flow injection analysis (FIA) Syringe pump delivery Automated valve switching for sample-reference bracketing Typical injection volume: 40 µL Infusion time optimized for signal stability Data Processing Workflow Step 1: Raw Data Conversion Thermo .raw files → IsoX software → .isox files Isotopologue-resolved ion current extraction Mass accuracy verification Peak detection and integration Step 2: R-based Statistical Pipeline Quality control and filtering: Outlier removal via AGC window filtering TIC×IT (Total Ion Current × Injection Time) thresholding Satellite peak flagging and exclusion Robust detection filtering (isotopologue presence >30% of scans) Ratio calculations: Block-wise aggregation with multiple methods: Sum of ion intensities Arithmetic mean Geometric mean Linear regression slope Base peak assignment (M0 or most abundant isotopologue) Delta value calculations: Drift correction via bracketing standards Reference-sample block alternation Full error propagation: Standard error of the mean (SEM) Shot noise estimates based on counting statistics Combined uncertainty propagation Statistical analysis: Principal Component Analysis (PCA) Data centering and scaling Eigenvalue decomposition Variance explained calculations ANOVA for variable selection Welch's test for groups with unequal variances Post-hoc pairwise comparisons Significance testing (α = 0.05) Quality Control Measures Replicate injections: n=3 per manufacturer per run, n=3 analytical runs (total 9 replicates per manufacturer) Blank subtraction: Solvent blanks between sample injections Bracketing standards: Reference material injections before and after each sample Incomplete scan filtering: Removal of scans missing isotopologue signals Resolution validation: Peak resolution monitoring for enhanced Fourier Transform (eFT) consistency Mass accuracy: Continuous monitoring of measured vs. theoretical m/z values FAIR Principles Compliance Findable ✓ Unique identifier: Zenodo-assigned DOI ✓ Rich metadata: Comprehensive descriptions of experimental design, instruments, and methods ✓ Keywords: Isotopologue profiling, Orbitrap mass spectrometry, Imidacloprid, Source attribution, Compound-specific isotope analysis (CSIA), Manufacturer fingerprinting, Isotope hydrology, Natural abundance, High-resolution mass spectrometry, Pesticide forensics Accessible ✓ Open access: CC-BY-4.0 license (or specify your license) ✓ Long-term preservation: Zenodo repository with CERN infrastructure ✓ Standard formats: .raw (Thermo native format) .isox (tab-delimited text) .csv/.tsv (sequence/metadata) .R/.Rmd (analysis scripts) ✓ No authentication barriers: Publicly downloadable Interoperable ✓ Industry-standard formats: Thermo RAW format compatible with: Thermo Xcalibur Thermo FreeStyle Viewer ProteoWizard MSConvert Other third-party MS tools ✓ Documented data structures: ISOX format fully described in Hilkert et al. (2021) Analytical Chemistry DOI: 10.1021/acs.analchem.1c00944 ✓ Cross-platform compatibility: R scripts tested on Windows, macOS, Linux ✓ Standard isotope nomenclature: IUPAC conventions for isotopologue notation Reusable ✓ Complete workflows: End-to-end processing from raw data to publication-ready results ✓ Detailed parameters: All processing settings documented in R Markdown reports ✓ Annotated code: Inline documentation explaining each processing step ✓ Provenance information: Script version tracking Processing date stamps Software dependencies listed ✓ External standards: Calibration to international scales (VPDB, Air, VSMOC) ✓ Clear licensing: Permissive license for data reuse Technical Specifications File Formats and Sizes RAW files: Thermo proprietary binary format (~50-200 MB per file) ISOX files: Tab-delimited text (~1-10 MB per file) CSV/TSV files: Comma/tab-separated values (<1 MB) R scripts: Plain text R code (.R, .Rmd) Software Requirements Data Acquisition Thermo Xcalibur (version 4.x or higher) Thermo Tune interface for instrument control Data Processing IsoX (Thermo Fisher Scientific): Isotopologue extraction from Orbitrap data Version: Compatible with Exploris/Q Exactive platforms Reference: Hilkert et al., Anal. Chem. 2021, DOI:10.1021/acs.analchem.1c00944 Statistical Analysis R (≥ 4.0.0) R packages: isoorbi: Dual-inlet Orbitrap workflows tidyverse: Data manipulation (dplyr, ggplot2, tidyr, readr) openxlsx: Excel report generation ggpubr: Publication-ready plots ggrepel: Label positioning in plots gridExtra: Multi-panel plot arrangements progress: Progress bar utilities File Structure Summary Rawdata_for_Publication/ ├── Manufacturer_Fingerprinting/ │ ├── MS1/ │ │ ├── RAW/ # Thermo RAW files (Exploris 240) │ │ ├── data/ │ │ │ ├── *.isox # 25 ISOX files │ │ │ ├── isotopologs.tsv # Target isotopologue definitions │ │ │ ├── sequence.csv # Sample metadata │ │ │ └── Referenciation.csv # Reference standards │ │ └── flow_injection_w_syringe_M0-V4_AS_Calibration.Rmd │ ├── MS2/ │ │ ├── RAW/ # Thermo RAW files (ddMS²) │ │ ├── Fragment 84/data/ # Fragment-specific ISOX + metadata │ │ ├── Fragment 175/data/ # Fragment-specific ISOX + metadata │ │ └── Fragment 209/data/ # Fragment-specific ISOX + metadata │ └── R_Scripts_PCA/ │ ├── 20251014_One_Way_Anova_Welch_Data_Revised_3.R │ ├── 20251014_PCA_Rework_Final_Rescale_4_Dataset_1.R │ └── 20251014_PCA_Rework_Final_Rescale_4_Dataset_2.R └── Imidacloprid_Hydrolysis/ ├── combined.isox # Time-series isotopologue data ├── isotopologs.tsv # Target definitions └── 20250623_IsoOrbi_Dua-Inlet_AutoPath_AutoEval_V7.R Total file count: ~100+ files Estimated total size: ~10-20 GB (depending on RAW file inclusion) Notes for Data Users Getting Started Download the dataset from Zenodo Extract RAW files (if analyzing from scratch) or use provided ISOX files Install R and required packages (see Software Requirements) Run R Markdown scripts to reproduce analyses Consult inline documentation in scripts for parameter customization Common Use Cases Source attribution: Use MS1 and MS2 PCA results to compare unknown samples to manufacturer fingerprints Method development: Adapt R workflows for other pesticides or isotopologue studies Educational purposes: Demonstrate isotopologue profiling with Orbitrap MS Meta-analysis: Combine with other CSIA datasets for broader studies Troubleshooting ISOX file reading issues: Ensure tab-delimited format is preserved R package errors: Check package versions and dependencies Memory limitations: Large RAW files may require 16+ GB RAM for processing Platform compatibility: Scripts developed on Windows; paths may need adjustment for macOS/Linux