DOI: 10.5281/zenodo.17592128
title: Manufacturer-Specific Isotopologue Fingerprints and Hydrolysis Kinetics of Imidacloprid: High-Resolution Orbitrap Mass Spectrometry Data and Statistical Analysis

description: 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