High-Resolution Mass Spectrometry for Nitrate Aerosol Isotopologue Quantification: Method Development, Calibration, and Application to Atmospheric Samples

Variations in the stable isotope ratios of nitrate (NO₃^-) provide powerful constraints on atmospheric oxidation chemistry and source apportionment; however, traditional isotope ratio mass spectrometry (IRMS) methods require large sample sizes (≥50 nmol) and specialized setups and cannot access multiply substituted ("clumped") isotopologues. We present a new high-resolution electrospray ionization Orbitrap mass spectrometry (ESI-Orbitrap) approach for direct quantification of NO₃^- isotopologues paired with an optimized preparation workflow to minimize matrix- and concentration-dependent biases. Accurate isotopic measurement was highly sensitive to sample concentration and coextracted solutes, with deviations up to 3-5‰ in δ¹⁸O and δ¹⁷O for unpurified or dilute (<25 μM) solutions. These effects were mitigated by ion chromatography (IC) purification, automated NO₃^- fraction collection, neutralization with equimolar NaOH, controlled water removal, and methanol reconstitution at ∼100 μM. Calibration against USGS34 and USGS35 standards yielded precise δ¹⁵N, δ¹⁸O, and Δ¹⁷O values (±0.4-0.6‰, ± 1σ) and enabled characterization of clumped NO₃^- isotopologue values (Δ¹⁵N¹⁸O, Δ¹⁷O¹⁸O, Δ¹⁸O¹⁸O). Aerosol samples showed excellent agreement with the bacterial denitrifier-IRMS method for δ¹⁵N (<i>R</i>² = 0.891, slope = 1.3 ± 0.1, mean bias = -0.3‰) and Δ¹⁷O (<i>R</i>² = 0.786, slope = 1.10 ± 0.1, mean bias = 0.5‰) and a modest offset for δ¹⁸O (<i>R</i>² = 0.774, slope = 0.8 ± 0.1, bias = -3.3‰). The resulting isotopic ranges (δ¹⁵N = -3 to +8‰; Δ¹⁷O = 18-28‰) align with midlatitude aerosol observations. Preliminary stochastic-normalized clumped isotope data reveal distinct behaviors among aerosol nitrate isotopologues, with Δ¹⁷O¹⁸O being sensitive to oxidation chemistry, Δ¹⁸O¹⁸O largely decoupled from bulk oxygen isotope tracers, and Δ¹⁵N¹⁸O integrating both nitrogen source and oxidative process information. This method expands isotopic constraints for atmospheric NO₃^- and establishes a foundation for multi-isotopologue analyses of other oxyanion aerosols.