Method for Small-Molecule Discovery Based on Microscale


Method for Small-Molecule Discovery Based on Microscale...

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Anal. Chem. 2008, 80, 6293–6299

Method for Small-Molecule Discovery Based on Microscale-Preparative Multidimensional Gas Chromatography Isolation with Nuclear Magnetic Resonance Spectroscopy Graham T. Eyres,† Sylvia Urban,‡ Paul D. Morrison,† Jean-Pierre Dufour,§,| and Philip J. Marriott*,† Australian Centre for Research on Separation Science (ACROSS) and Marine And Terrestrial Natural Product (MATNAP) Research Group, School of Applied Sciences, RMIT University, G.P.O. Box 2476V, Melbourne, Victoria 3001, Australia, and Department of Food Science, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand Absolute chemical identification requires obtaining a pure compound followed by structure elucidation using spectroscopic techniques, principally NMR spectroscopy and mass spectrometry. Classical isolation techniques suffer from insufficient resolution for complex samples, requiring time-consuming fractionation in multiple steps. Here, a novel preparative technique based upon capillary column multidimensional gas chromatography (MDGC) with 2D NMR to resolve, isolate, and identify pure volatile components from a complex sample is described. As a model application, geraniol was isolated from an essential oil matrix using MDGC and quantitatively resolved from 15 partially coeluting compounds from the first column. Geraniol was recovered from 10 (8.6 µg) and 100 injections (77.6 µg; purity >99%) for subsequent NMR analysis at 500 and 800 MHz (with cryoprobe). Proton and gCOSY NMR experiments were successfully performed at 12.3 µg/mL (10 injections), while gHSQC and gHMBC NMR experiments were obtained at 110.8 µg/ mL (100 injections). This approach is applicable to the biodiscovery of volatile molecular species or, indeed, any volatile compound in a complex matrix that requires confirmation of component identity. Gas chromatography (GC) and mass spectrometry (MS) are indispensable for identification of volatile compounds. Difficulties arise when compounds cannot be unambiguously identified by their mass spectra and retention data alone. This may be due to their absence from mass spectral databases, high similarities between isomeric compounds, or absence of the molecular ion. For instance, sesquiterpenoids are notoriously difficult to resolve, exhibiting very similar mass spectra,1 and this problem is far from being an isolated case. * To whom correspondence should be addressed. Tel.: +61 3 99252632. Fax: +61 3 99253747. E-mail: [email protected]. † Australian Centre for Research on Separation Science (ACROSS), RMIT University. ‡ Marine And Terrestrial Natural Product (MATNAP) Research Group, RMIT University. § University of Otago. | Deceased 26 February 2007. (1) Ko ¨nig, W.; Bu ¨ low, N.; Saritas, Y. Flavour Frag. J. 1999, 14, 367–378. 10.1021/ac8007847 CCC: $40.75  2008 American Chemical Society Published on Web 07/23/2008

Absolute chemical identification requires compounds of high purity, for characterization by a combination of spectroscopic techniques, principally nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. In the absence of pure commercially available compounds, they must be obtained by either isolation from a natural product extract, or by synthesis. At present, NMR spectroscopy of volatile compounds is typically performed off-line. Pure compound isolation can be timeconsuming and tedious using preparative chromatographic procedures for sample fractionation, with the risk of the compound being lost, altered, or contaminated. Consequently, online liquid chromatography (LC) hyphenated to NMR has become a valuable technique.2–4 While online GC-NMR has been recently demonstrated,5 it is exceedingly difficult to implement requiring significant alterations to the NMR instrument, e.g., use of a specialized microprobe, and has limited chromatographic and spectroscopic resolution and sensitivity. Although Grynbaum and co-workers5 presented promising results for the concept of online GC-NMR, it remains unrealistic for routine analysis of complex samples. Preparative GC has often been used as a final step to isolate pure volatile compounds. Historically, packed columns have been useful because of their high loading capacity, but resolution of peaks in complex samples remains poor. More recently, widebore (0.32 mm) or megabore (0.53 mm) capillary columns have yielded better resolution,6–10 but frequent coelution of compounds in single-column GC (1DGC) still arises.11 The necessity for two-dimensional separations is well documented.11–14 Peaks are neither evenly nor randomly distributed (2) Urban, S.; Separovic, F. In Frontiers in Drug Design and Discovery; Caldwell, G. W., Rahman, A.-u., Springer, B. A., Eds.; Bentham Science Publishers: Hilversum, The Netherlands, 2005; Vol. 1, pp 113-166. (3) Albert, K., Ed. On-line LC-NMR and Related Techniques; John Wiley & Sons: New York, NY, 2002. (4) Exarchou, V.; Krucker, M.; van Beek, T. A.; Vervoort, J.; Gerothanassis, I. P.; Albert, K. Magn. Reson. Chem. 2005, 43, 681–687. (5) Grynbaum, M. D.; Kreidler, D.; Rehbein, J.; Purea, A.; Schuler, P.; Schaal, W.; Czesla, H.; Webb, A.; Schurig, V.; Albert, K. Anal. Chem. 2007, 79, 2708–2713. ¨ .; Andersson, (6) Holmstrand, H.; Mandalakis, M.; Zencak, Z.; Gustafsson, O P. J. Chromatogr., A 2006, 1103, 133–138. (7) Mandalakis, M.; Gustafsson, Ö. J. Chromatogr., A 2003, 996, 163–172. (8) Eglinton, T. I.; Aluwihare, L. I.; Bauer, J. E.; Druffel, E. R. M.; McNichol, A. P. Anal. Chem. 1996, 68, 904–912.

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in a chromatogram because compounds often have related chemical or physical properties.15 System peak capacity must be much greater than the number of compounds in the sample,11,12 and considerable coelution of peaks in complex samples is inevitable in 1DGC. Comprehensive two-dimensional GC (GC × GC) reveals the enormous complexity that is possible for complex samples, as demonstrated for atmospheric particulate samples.16 In a recent study, an important odorant was determined to coelute with up to 13 compounds in a fractionated hop essential oil sample, and only multidimensional GC could adequately resolve this compound.17 Preparative multidimensional gas chromatography (prepMDGC) with capillary columns has been previously demonstrated in the literature.18–22 Previous applications employing prep-1DGC for isolation of volatile compounds will be improved through use of the present prep-MDGC system. For example, Meinert et al.9 collected 11 fractions from 600 injections of a p-nonylphenol sample over a 15-day period (run time of each analysis 28.7 min). The fractions were reinjected into GC/MS for identification, but considerable coelution was evident with at least seven peaks eluting in one fraction. Prep-MDGC will generate far superior purity. In another study, prep-1DGC, mass spectrometry, and 1H NMR was recently used to identify an important sex pheromone of the German cockroach with implications for pest control and disease prevention.23,24 Pheromone isolation required many preparative steps, including flash chromatography of a total lipid extract into 6 fractions, then HPLC into 39 fractions, with each fraction tested to determine biological activity. Finally, the pure compound (0.05-0.5 µg) was collected by preparative 1DGC with a single injection. Capillary prep-MDGC, would significantly reduce or eliminate altogether the lengthy sample preparation. Capillary prep-MDGC should also improve isolation of polyaromatic hydrocarbons for radiocarbon dating, where the 1DGC application required 60 injections (run time >60 min each) onto a 60 m × 0.53 mm i.d. column.25 (9) Meinert, C.; Moeder, M.; Brack, W. Chemosphere 2007, 70, 215–223. (10) Bicchi, C.; D’Amato, A.; Manzin, V.; Galli, A.; Galli, M. J. High Resolut. Chromatogr. 1997, 20, 493–498. (11) Davis, J. M.; Samuel, C. J. High Resolut. Chromatogr. 2000, 23, 235–244. (12) Davis, J. M.; Giddings, J. C. Anal. Chem. 1983, 55, 418–424. (13) Giddings, J. C. J. Chromatogr., A 1995, 703, 3–15. (14) Giddings, J. C. Anal. Chem. 1984, 56, 1258A–1264A. (15) Marriott, P. J.; Kinghorn, R. M. In Gas Chromatographic Techniques and Applications; Handley, A. J., Adlard, E. R., Eds.; Sheffield Academic Press: Sheffield, England, 2001; pp 260-297. (16) Lewis, A. C.; Carslaw, N.; Marriott, P. J.; Kinghorn, R. M.; Morrison, P.; Lee, A. L.; Bartle, K. D.; Pilling, M. J. Nature 2000, 405, 778–781. (17) Eyres, G. T.; Marriott, P. J.; Dufour, J.-P. J. Agric. Food Chem. 2007, 55, 6252–6261. (18) Nitz, S.; Drawert, F.; Albrecht, M.; Gellert, U. J. High Resolut. Chromatogr. 1988, 11, 322–327. (19) MacNamara, K.; Hoffmann, A. In Instrumental Methods in Food and Beverage Analysis; Wetzel, D., Charalambous, G., Eds.; Elsevier Science: Amsterdam, The Netherlands, 1998; pp 303-346. (20) Nitz, S.; Kollmannsberger, H.; Drawert, F. J. Chromatogr., A 1989, 471, 173–185. (21) Rijks, J. P. E. M.; Rijks, J. A. J. High Resolut. Chromatogr. 1990, 13, 261– 266. (22) Werkhoff, P.; Guntert, M.; Krammer, G.; Sommer, H.; Kaulen, J. J. Agric. Food Chem. 1998, 46, 1076–1093. (23) Nojima, S.; Kiemle, D. J.; Webster, F. X.; Roelofs, W. L. J. Chem. Ecol. 2004, 30, 2153–2161. (24) Nojima, S.; Schal, C.; Webster, F. X.; Santangelo, R. G.; Roelofs, W. L. Science 2005, 307, 1104–1106.

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Important improvements in instrumentation for GC have led to improved retention time reproducibility, making capillary prepMDGC a viable approach to isolate pure compounds from complex mixtures. Once isolated, the appropriate spectroscopic method(s) must be selected for identification. Mass spectrometry is an obvious choice, but is still insufficient for many compounds, e.g., isomers. Recent developments in NMR such as LC-NMR, the use of increasingly sensitive probes, and higher field-strength magnets suggest that it should be re-evaluated as a means for structure determination when combined with GC. The development of microscale-preparative MDGC is proposed here to completely resolve and isolate compound(s) of interest from a complex matrix with minimal prior fractionation. Trapping the isolated compound during the course of multiple injections produces sufficient quantities to facilitate off-line NMR spectroscopic analysis, with positive identification achieved in combination with mass spectrometry. This study is the first to demonstrate 2D NMR on a microscale-preparative sample isolated from a GC separation and can be considered generally applicable to all volatile compounds. EXPERIMENTAL SECTION Samples and Solvents. Geraniol (1) (∼98%) was purchased from Aldrich Chemical Co. (St. Louis, MO). Sample solutions for GC were prepared using n-hexane (Pestanal, g95%; Riedel-de-Hae¨n,

Seelze, Germany). Deuterated methanol (CD3OD, UVAsol 99.8%; Merck KgaA, Darmstadt, Germany) was used for NMR spectroscopy; geraniol was found to be unstable in deuterated chloroform (CDCl3, 99.8% stabilized with silver and 0.1% deuterated pyridine; Cambridge Isotope Laboratories, Inc., Andover, MA) at concentrations of