Nitrogen-containing species in bio-oils obtained from fast pyrolysis of switchgrass were studied using high resolution mass spectrometry at various harvest times throughout the year. Almost three hundred chemical compositions of nitrogen species were determined through efficient ionization and accurate mass information. N-2 is the most abundant heteroatom class, followed by NO, N2O, NO2, and N-1 compounds. Nitrogen species, especially N-2 compounds, dominate the bio-oil spectra in early summer, but decrease significantly in later harvest times. From the contour plots of double bond equivalent versus carbon number and tandem mass spectrometric analysis, the major structural motif for N-1 and NO class compounds are assigned as pyridine and that of N-2 class compounds as imidazole. The dramatic decrease of N-2 class compounds in delayed harvest bio-oils is well correlated with the decomposition of proteins, represented by imidazole as a pyrolysis product of histidine, as the senescence of the perennial plant proceeds. Some of the heterocyclic aromatic compounds are also found in gas chromatography-mass spectrometry, further supporting our analysis. (C) 2013 Elsevier Ltd. All rights reserved.