Oxyanion Isotopologue Analysis of Natural Samples by Electrospray-Orbitrap: Method, Validation and Clumped Isotopes

Traditional gas-source, sector-field isotope ratio mass spectrometers are not optimally designed to analyse non-volatile molecular solutes such as oxyanions or organics. Tedious preparation steps – often requiring micromole-scale samples – break chemical bonds, meaning only elemental isotope ratios can be extracted. Recent advances in mass spectrometry point towards a novel isotopic technique: soft electrospray ionisation coupled to a high-resolution Orbitrap mass spectrometer (ESI-Orbitrap). Widely accessible and commonly used for bioanalysis, this combination permits high mass resolution analysis on intact molecules in liquid solutions, currently requires tens of nanomoles of sample, provides simultaneous multi-isotopologue analysis, and – in theory – permits clumped isotope measurements. In this study, we (i) present procedures for the preparation of natural aqueous samples for ESI-Orbitrap, nitrate and sulphate isotopologue analysis and subsequent data treatment; (ii) provide improved ESI-Orbitrap stability metrics; (iii) validate ESI-Orbitrap performance for nitrate and sulphate singly-substituted isotopologues in natural samples, relative to traditional isotopic methods; (iv) report measurements of previously inaccessible nitrate clumped isotopes; and (v) discuss the present limitations of ESI-Orbitrap for isotopologue analysis and avenues for optimisation. We find that ESI-Orbitrap exhibits matrix sensitivity and isotopic drift over periods exceeding 30 minutes. However, we demonstrate that careful purification of ice core and aerosol samples, application of sample-reference bracketing and performance of linear calibrations allow accurate ESI-Orbitrap measurements of δ15N-NO3-, δ18O-NO3-, Δ17O-NO3-, δ34S-SO42-, and Δ17O-SO42-, achieving 0.4 – 1.3 ‰ precision while consuming only 20 nmol nitrate or 10 nmol sulphate. Using ESI-Orbitrap no-M0 analysis, we also measure the previously-inaccessible nitrate clumped isotope values Δ15N18O-NO3- and Δ18O18O-NO3- in ice core samples with uncertainties of 2.3 ‰ and 3.3 ‰, respectively. We outline avenues for improving the sensitivity, accuracy and precision of ESI-Orbitrap isotopologue measurements, and consider how our method could be adapted to additional analytes in different sample matrices.