loading . . . Accurate Quantification of 45N2O with a Membrane Inlet Mass Spectrometer by Optimizing the Temperature of a Cold Trap Membrane Inlet Mass Spectrometry (MIMS) is extensively applied in the study of nitrogen cycling processes. However, robust methods for the quantitative detection of N2O by this technique are still scarce. In this study, acidified azide was employed to reduce 15NO2ā to generate 45N2O, which was used to make a standard curve for qualifying 45N2O with MIMS. Key parameters affecting the azide reduction, including reagent concentration, reaction time, reaction mode, and salinity, were systematically evaluated and optimized. Then, the generated 45N2O dissolved in water was introduced into the MIMS via a cold trap maintained at ā88 ± 2 °C. This specific temperature capitalizes on the difference in the freezing points of N2O (ā91 °C) and CO2 (ā78 °C), thereby effectively minimizing CO2 interference during N2O measurement. Under optimized conditions, the linear fitting between 45N2O ion current intensity and expected 45N2O concentration showed an excellent linearity in the range of 0.1ā100 μmol/L (R2 = 0.998), with a detection limit of 0.1 μmol/L. This method achieves accurate (relative error: 4.688%) and precise (relative standard deviation: 1.560%) detection of 45N2O and holds potential for future application in tracing microbial N2O sourceāsink processes across diverse habitats. https://pubs.acs.org/doi/full/10.1021/jacsau.5c01146