Stable isotope analysis of Neonicotinoids by LC-IRMS and Orbitrap IRMS: instrumental challenges, method development, and environmental transformation

The relative abundances of stable isotopes in organic molecules (e.g., 13C/12C, 15N/14N) differ at the permil level. Stable isotope analysis (SIA) is used to resolve these differences, enabling their use as tracers. The isotopic composition of industrial chemicals depends on source materials and synthesis pathways used by manufacturers. Additionally, characteristic isotope fractionation caused by environmental transformation (e.g., hydrolysis or photolysis) can be investigated by SIA, providing mechanistic insight. In this thesis, this is exemplified for neonicotinoid insecticides that have raised ecological concerns due to their adverse effects on pollinators and aquatic invertebrates. The aim of this thesis was to advance compound- and fragment-specific SIA instrumentation and methodology for neonicotinoids. The performance of liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) for 13C/12C analysis of polar, thermally labile neonicotinoids was evaluated. Shortcomings of the wet persulfate oxidation interface were identified, with recalcitrance and isotope-ratio bias observed for guanidine/nitroguanidine moieties of neonicotinoids. Practical strategies for assessing and mitigating such problems were formulated. An attempt to improve reactor design by combining UV and thermal persulfate activation did not yield superior performance. An LC-IRMS/HRMS workflow with a post-column splitter was developed to provide simultaneous 13C/12C ratios and Orbitrap high-resolution mass spectra. Imidacloprid (IMI) was hydrolyzed and photolyzed in batch experiments, and carbon isotope enrichment factors (εC) were distinguished across processes: negligible under UV-C (254 nm), small under simulated sunlight, and largest during alkaline hydrolysis at pH 12 and 30 °C (εC = -3.9±0.4‰); product analysis indicated a single reactive carbon, allowing calculation of an apparent site-specific carbon enrichment factor (εC = -34.1±3.7‰). Direct isotope ratio measurements by ESI-Orbitrap IRMS at natural abundance were used for fingerprinting seven IMI manufacturers. Intact molecules were analyzed without a conversion interface, enabling multi-element (C, N, Cl) and multiple-substitution information, and MS2 capabilities provided fragment-specific isotope ratios. These multidimensional isotopic datasets were exploited by principal component and cluster analysis, with variable selection used to either maximize discrimination or to reveal relationships indicative of shared source materials or synthesis pathways between manufacturers. During alkaline hydrolysis (30 °C, pH 12), carbon and nitrogen enrichment factors were determined by ESI-Orbitrap IRMS as εC = -4.3±1.4‰ and εN = 0.6±1.6‰, complementing the prior LC-IRMS result for εC and supporting a carbon-centered, rate-limiting step. Overall, this thesis demonstrates the ability of SIA to disentangle the sources and transformation pathways of IMI. LC-IRMS is expected to advance through improvements to the oxidation interface. It also demonstrates the untapped potential of Orbitrap IRMS as a sensitive, multi‑element, compound‑ and fragment‑specific alternative.