Toward Molecular Forensics of Agrochemicals with Orbitrap IRMS: Isotopic Fingerprints of Imidacloprid Sources and Elucidating Hydrolysis

Imidacloprid (IMI) is a widespread neonicotinoid insecticide of environmental concern because of its ecotoxicity and persistence. Tracing its commercial sources and environmental transformation is difficult with concentration-based approaches. We explored electrospray ionization Orbitrap isotope ratio mass spectrometry ((ESI) Orbitrap MS-Based Isotope Ratio Analysis) for compound- and fragment-specific stable isotope analysis of IMI at natural abundance. This approach enables direct measurement of ¹³C, ¹⁵N, ³⁷Cl, and multiple substituted isotopologue ratios. Experimental parameters were optimized to control sources of bias in isotopologue ratios. A dual-inlet bracketing protocol enabled normalization and drift correction. IMI from seven different commercial sources could be distinguished by their isotopic fingerprints. We demonstrate that principal component analysis (PCA) can exploit the expanded set of isotopic variables. Two PCAs with distinct variable sets were performed: a fragment-only, nonoverlapping isotopologue subset for source material attribution, and a combined molecular-average and fragment-level subset to maximize manufacturer discrimination. Alkaline hydrolysis (pH ≈ 12, 30 °C) based on compound-average δ¹³C and δ¹⁵N values measured by (ESI) Orbitrap MS-Based Isotope Ratio Analysis revealed significant carbon isotope fractionation (εC = -4.3 ± 1.4 ‰) and a statistically negligible nitrogen fractionation (εN = 0.6 ± 1.6 ‰), consistent with initial OH⁻ attack at the nitroimine carbon as the primary pathway. (ESI) Orbitrap MS-Based Isotope Ratio Analysis can elucidate transformation mechanisms in analytically challenging polar analytes. The chemometric approach of using MS¹/MS² data can be transferred to other (agro-)chemicals either to enhance isotopic discrimination or to discover patterns.