Quantification of hydrazine in biochemical assays and anammox bacteria using LC-MS

Abstract Hydrazine is an industrially valuable product. Strikingly, hydrazine is also a key intermediate in the energy metabolism of anaerobic ammonium-oxidizing (anammox) bacteria, where it is formed by hydrazine synthase. To study the molecular mechanism and activity of isolated hydrazine synthase, a sensitive, relatively fast and easy method to quantify hydrazine is needed. However, reported methods such as colorimetric assays, MALDI-TOF MS, and enzymatic conversion of hydrazine to dinitrogen gas, are either insensitive or laborious. In this study, we describe the validation and application of a fast and simple liquid chromatography-mass spectrometry (LC-MS) method to reproducibly quantify hydrazine produced by anammox hydrazine synthase. Hydrazine was derivatized with benzaldehyde, and directly injected onto a C18 column coupled to a Q-TOF MS. To increase assay performance, 15 N 2 -hydrazine was included as internal standard. The response ratio of hydrazine was linearly proportional to the hydrazine concentration from 0.05-1 µM with an average correlation coefficient of 0.9925. Intra- and inter-day accuracy lay between 88-113% and 95-105%, respectively. Intra- and inter-day precision (RSD, %) ≤ 11%. Hydrazine and derivatized hydrazine were stable when stored at -70°C or in the autosampler. We successfully applied the LC-MS method to determine hydrazine production by isolated hydrazine synthase and within cell lysate of anammox bacteria.