A compound-specific isotope method for measuring the stable nitrogen isotopic composition of tetrapyrroles Yoshito Chikaraishi a, * , Yuichiro Kashiyama a , Nanako O. Ogawa a , Hiroshi Kitazato a , Mitsuru Satoh b , Shinya Nomoto b , Naohiko Ohkouchi a a Institute for Research on Earth Evolution, Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima-cho, Yokosuka 237-0061, Japan b Department of Chemistry, University of Tsukuba, Tsukuba 305-8571, Japan Received 22 February 2007; received in revised form 15 August 2007; accepted 24 August 2007 Available online 5 September 2007 Abstract The stable nitrogen isotopic signature of natural tetrapyrroles such as chloro- and pheo-pigments and alkylporphyrins is a potentially valuable proxy for the reconstruction of the nitrogen cycle of modern aquatic environments and of the geo- logical past. However, its application has been limited due to analytical difficulties. In this study, we developed a method to determine stable nitrogen isotopic composition of tetrapyrroles by a combination of chemical conversion into monopyrrole units (i.e. maleimides) and their isotope analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Two authentic (pyropheophorbide a and mesoporphyrin IX methylester) and four natural (chlorophyll a, deoxophylloerythroetioporphyrin (DPEP), and two 17-nor-DPEP) tetrapyrroles were converted to maleimides by HCl treatment and chromic acid oxidation. The stable nitrogen isotopic composition of the maleimides can be determined by GC/C/IRMS, with a standard deviation (1r) of better than ± 0.5& and the minimum sample amount of 0.8 nmol N. The isotopic composition of the maleimides is consistent with that of the original tetrapyrroles, suggesting nitrogen isoto- pic fractionation during the chemical treatment was insignificant. Further, no substantial difference is observed in the iso- topic composition among the maleimides derived from a single tetrapyrrole. The developed method is applicable for the nitrogen isotope analysis of natural tetrapyrroles in various biological and geological samples. Ó 2007 Elsevier Ltd. All rights reserved. 1. Introduction Since chloropigments such as chlorophylls and bacteriochlorophylls are representatives of natural tetrapyrroles directly related to the photosynthetic processes of photoautotrophs (e.g. Blankenship, 2002; Grimm et al., 2006), they are definitive bio- markers of photoautotrophic activity in the aquatic surface environment (e.g. Sanger, 1988; Nakajima et al., 2003). Moreover, the chloropigments are long preserved as pheo-pigments such as pheophytins and bacteriopheophytins in oceanic and lacustrine sedi- ments (e.g. Sanger, 1988; Keely et al., 1990; Prowse and Maxwell, 1991; Airs et al., 2001; Squier et al., 2002; Tolosa et al., 2004) and as metallo-alkylpor- phyrins such as VO deoxophylloerythroetioporphy- 0146-6380/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.orggeochem.2007.08.010 * Corresponding author. Tel.: +81 46 867 9778; fax: +81 46 867 9775. E-mail address: ychikaraishi@jamstec.go.jp (Y. Chikaraishi). Available online at www.sciencedirect.com Organic Geochemistry 39 (2008) 510–520 www.elsevier.com/locate/orggeochem Organic Geochemistry