RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2006; 20: 926–932 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.2386 From mass to structure: an aromaticity index for high-resolution mass data of natural organic matter B. P. Koch1* and T. Dittmar2 1Alfred-Wegener-Institut fu¨r Polar- und Meeresforschung, Am Handelshafen 12, 27570 Bremerhaven, Germany 2Florida State University, Department of Oceanography, OSB 311, Tallahassee, FL 32306-4320, USA Received 16 November 2005; Revised 9 January 2006; Accepted 9 January 2006 Recent progress in Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) has provided extensive molecular mass data for complex natural organic matter (NOM). Structural information can be deduced solely from the molecular masses for ions with extreme molecular element ratios, in particular low H/C ratios, which are abundant in thermally altered NOM (e.g. black carbon). In this communication we propose a general aromaticity index (AI) and two threshold values as unequivocal criteria for the existence of either aromatic (AI > 0.5) or condensed aromatic structures (AI ‡ 0.67) in NOM. AI can be calculated from molecular formulae which are derived from exact molecular masses of naturally occurring compounds containing C, H, O, N, S and P, and is especially useful for substances with aromatic cores and few alkylations. In order to test the validity of our model index, AI is applied to FTICRMS data of a NOM deep-water sample from the Weddell Sea (Antarctica), a fulvic acid standard, and an artificial dataset of all theoretically possible molecular formulae. For graphical evaluation a ternary plot is suggested for four-dimensional data representa- tion. The proposed aromaticity index is a step towards structural identification of NOM and the molecular identification of polyaromatic hydrocarbons in the environment. Copyright # 2006 John Wiley & Sons, Ltd. Natural organic matter (NOM) is a highly complex and analysis of NOM.4–7 In combination with electrospray polydisperse mixture of different compounds,1 the number ionization (ESI), extensive molecular elemental information of which is still unknown but exceeds several thousands. It is can be achieved without preceding chromatographic separa- abundant in terrestrial, limnic and marine environments. tion. Due to its very high resolution (>200 000) and mass Major contributors to the global NOM pool are humic accuracy (often < 0.5 ppm), FTICRMS delivers unequivocal substances, sedimentary and dissolved organic matter, molecular formulae. Formula assignment is possible because which are operationally defined. Highly degraded NOM NOM consists of few abundant elements, mainly of C, H, and like dissolved organic matter (DOM) in the deep ocean resists O, and to a lower degree of N, P and S. Other elements further degradation and can reach average ages of several are rare in NOM and can be disregarded for a general thousand years.2 The extent of molecular information on examination of molecular structures in NOM. NOM is largely restricted due to its complexity which NOM is a very heterogeneous mixture of largely different prevents molecular resolution in chromatographic methods. molecules. Molecular O/C and H/C ratios range from 0–0.8 Also chemical degradation methods yield only small and 0.3–1.8, respectively, in both deep-sea DOM (Weddell amounts of analytically accessible compounds probably Sea, Antarctica)8 and a fulvic acid standard (Suwannee because of the refractory character of NOM. The proportion River).7,8 Typical molecular masses in DOM determined by of NOM that can be characterized on the molecular level is FTICRMS range from approximately 300 to 700 Da. Deduc- therefore small, in deep-sea DOM, e.g., less than 10% of tion of specific structural configurations exclusively from organic carbon can be assigned to molecular structures.3 a given molecular formula9–11 can be challenging. Even Fourier transform ion cyclotron resonance mass spectro- formulae of very small ions can result in a large variety of metry (FTICRMS) opened a new analytical window for the different structures and functionalities. In most cases structural information on NOM can only be achieved by *Correspondence to: B. P. Koch, Alfred-Wegener-Institut fu¨ r Polar- additional chemical and analytical techniques. However, for und Meeresforschung, Am Handelshafen 12, 27570 Bremerha- ven, Germany. molecules with extreme elemental ratios, the number of E-mail: [email protected] possible isomers is more restricted. Low H/C ratios, for Contract/grant sponsor: The Petroleum Research Fund; instance, diminish the number of configurational isomers contract/grant number: ACS PRF#41515-G2. Contract/grant sponsor: The National Oceanic and Atmospheric and can be associated with unsaturations and C–C double Administration; contract/grant number: NOAA GC05-099. bonds. The calculation of the ‘double-bond equivalent’ (DBE) Contract/grant sponsor: Deutsche Forschungsgemeinschaft; is a well-established tool in mass spectrometry. DBE contract/grant number: DFG KO 2164/3-1. Contract/grant sponsor: The German Academic Exchange Ser- represents the sum of unsaturations plus rings in a molecule. vice; contract/grant number: DAAD PPP USA 315/ab. Since triple bonds or cumulated double bonds are rare in Copyright # 2006 John Wiley & Sons, Ltd. Aromaticity index for natural organic matter 927 NOM, a high density of double bonds (or high DBE/C ratios) All detected ions (m/z window 300–600) were singly can be indicative for aromatic or even condensed aromatic charged. Once the exact masses of the molecules had been structures. Recent results in NOM research show that natural determined, their molecular formulae were calculated by samples can contain significant amounts of thermogenic arbitrarily combining any possible combination of atoms. For carbon, i.e. mainly condensed functionalized polyaromatic each detected mass every possible chemical formula in a hydrocarbons (PAHs)8,12–16 with H/C ratios of less than 0.5. 0.001 Da mass window was computed. The following The potential to unambiguously identify condensed elements (and number of atoms of each element) were aromatic structures in NOM from ultrahigh-resolution mass considered in the calculation: 12C (1–100), 1H (1–200), 14N spectrometry data is intriguing because it provides a (0–10), 16O (0–50), 23Na (0–1), and 13C (0–1). The rules and molecular tool to simultaneously identify a large number assumptions described in Koch et al.8 were applied to of polyaromatic compounds of thermogenic origin.14 To date exclude formulae which do not occur abundantly in NOM, in all available methods to determine this fraction of NOM are particular: DBE must be an integer value, H/C 2.2, based on operationally defined parameters associated with O/C 1.2 and N/C 0.5. After applying these rules, all significant artefacts.17 Thermogenic carbon is believed to detected masses could be assigned to one unambiguous play a major role in the global carbon cycle. It can originate chemical formula. The most abundant ions (signal-to-noise from burning of biomass and fossil fuels, generally termed ratio >20) were all nitrogen-free, consisting of 12C, 1H, black carbon,14 and from petrogenic sources such as coals and 16O. and hydrothermal vents.15 The objective of this study was to develop an unambig- Generation of an artificial data matrix uous parameter for the identification of aromatic and To allow validation on a more general basis, an artificial condensed aromatic structures from ultrahigh-resolution dataset was constructed which comprised all theoretical mass spectrometry data. We introduce an aromaticity index molecular formulae in the mass range from 400–500 Da (AI) which reflects C–C double-bond ‘density’ in a molecule containing the elements C, H, O and N. For this purpose, all and which can be calculated solely from the exact molecular masses between 400 and 500 Da in 0.001 Da steps were fed masses of single NOM compounds. In order to test the into the same software we used for molecular formula validity of this new index, we applied AI to FTICRMS data computation from real mass spectrometry data. The same from deep-sea DOM (Weddell Sea, Antarctica),8 a fulvic acid assumptions and rules described above were applied to standard (Suwannee River), and to a calculated matrix of all exclude rare or impossible formulae. The final dataset theoretically possible molecular formulae in a mass range comprised a total of 25 130 possible molecular formulae. from 400–500 Da. RESULTS AND DISCUSSION EXPERIMENTAL The first step towards the establishment of an unambiguous Sampling and mass spectrometry index for the existence of aromatic and condensed aromatic In order to establish and validate an aromaticity index (AI), moieties in a molecule is to assess the degree of unsaturation we used the extensive ultrahigh-resolution mass spectro- or the density of C–C double bonds in a molecule. The sum of metry data set on marine and terrigenous DOM published by rings and double bonds in each molecule or ‘double-bond Koch et al.8 For details on sampling and mass spectrometry equivalent’ (DBE) can be calculated from the number of refer to this publication. In brief, samples were taken from atoms (Ni) and the valence (Vi) of each element (i) according the abyssal ocean (3500–4600 m) of the Weddell Sea to Eqn. (1). Uncharged molecules have a DBE with an integer (Antarctica). DOM was isolated from the saline aqueous value. matrix via C18 solid-phase extraction (Varian Mega Bond i Pmax Elut) at pH 2, and eluted with methanol. The Suwannee River NiðVi À 2Þ fulvic acid standard was obtained from the International DBE ¼ 1 þ i (1) Humic Substances Society (IHSS). For ESI, an aliquot of the 2 DOM methanol extract, or a methanolic solution of the IHSS By including common elements of NOM (C, H, O, N, S, P) standard, was mixed with Milli-Q water (50:50 v/v) and and their prevailing covalences, the DBE for NOM can be formic acid (0.2% final concentration).