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Indole Alkaloid Separation Using the Discovery® HS F5

By: Ilia Brondz, Klaus Høiland, David Bell,Amy R. Annino, Reporter EU Volume 23

Chemotaxonomic Study of Two Closely Related Brown-Spored Mushrooms

Ilia Brondz1, Klaus Høiland2, David Bell3 and Amy R. Annino3

1University of Life Sciences, Department of Chemistry, Biotechnology and Food Science, P.O. Box 5003, N-1432 Ås, Norway,e-mail: ilia.brondz@umb.no 
2 Department of Biology, University of Oslo, Norway 
3 Supelco, Bellefonte, Pennsylvania USA, e-mail:
aannino@sial.com

Abstract

Chromatographic analysis can be an extremely valuable tool in the taxonomic classifi cation of microorganisms, plants and fungi. In this study, the Discovery HS F5 column was used to analyze the indole alkaloid content of two closely related species of brown-spored mushroom: Cortinarius infractus and C. subtortus. The comparative indole alkaloid content analysis, along with chromatographic fi ngerprinting evidence, allowed the researchers to classify C. infractus and C. subtortus into two different taxonomical sections.

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Introduction

The pentafluorophenylpropyl phase of the Discovery HS F5 column offers excellent separation of alkaloid compounds under conditions suitable for LC-MS. The fluorinated stationary phase provides a sufficient substrate for ionic interactions with basic compounds so that ion pair reagents and phosphate buffers are not necessary. The aim of this investigation was to determine the sectionlevel taxonomic classification of two brown-spored fungi (C. infractus and C. subtortus, in the order Agaricales, Basidiomycota, subgenus Phlegmacium). Some researchers classify them into one section, Amarescens Mos., based on certain shared physical characteristics and partial DNA sequence information (1), while others place them into two sections, Infracti and Subtorti (5), based on several physical differences and supposed dissimilar alkaloid composition using bitter taste as evidence for the latter.

C. infractus has an unpleasant, fishy odor and a strong bitter taste which may partly be caused by the previously demonstrated presence of the indole alkaloids infractine, 6-hydroxyinfractine and infractopicrine (2). There is no known published chemical analysis for C. subtortus; however, this pleasant-smelling species with only a weakly bitter taste is not expected to contain indole alkaloids. As infractine-like alkaloids, which belong to the indole alkaloid group, may be exclusively found in C. infractus, these compounds may prove to have profound value as chemotaxonomic markers in the genus Cortinarius.

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Experimental

The Discovery HS F5 HPLC column was conditioned specifically for Supercritical Fluid (SFC) by flushing with 100 % methanol followed by a gradient of methanol: hexane 2 % increase per minute and subsequently held at 100 % hexane for 10 min. Before use, the column was flushed with carbon dioxide in the supercritical state.

SFC-MS analyses were performed on a Berger SFC MiniGram equipped with an UV K-2501 detector and ProNTo software. The flow stream exiting the UV detector was diverted by a fixed splitter and used to feed a Micromass PLCZ 4190 mass spectrometer equipped with ESI running under MassLynx. The biological samples were prepared as follows: 1 g samples of C. infractus and C. subtortus were air dried and extracted in 10 mL ethanol.

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Results and Discussion

The total ion chromatograms (TIC) for the extracts of C. infractus and C. subtortus are shown in Figures 1a and 1b, respectively. There are five significant peaks in the chromatogram of C. infractus, and four in the chromatogram for C. subtortus (almost all of which retain longer than the peaks for C. infractus). A mixture of the two extracts yielded no common peaks.

The SFC-MS fragmentation patterns obtained offered vital information to the identification of two compounds in the C. infractus extract: that which eluted at 4.6 min and that which eluted at 5.3 min (Figure 1a). It was proposed that the compound eluting at 4.6 min was infractopicrine based on the fragmentation pattern (which indicates that the compound in question contains fused aromatic rings) and molecular mass M+ detected was m/z 261. The structure of infractopicrine is shown in Figure 2a.


Figure 1. Chromatograms for C. infractus and C. subtortus(567517-U)



The peak that eluted at around Rt 5.3 min was putatively identified as pre-infractine. Though the poor fragmentation of this peak at a 60 V cone voltage, the [M+H]+ ion at m/z 241 was easily visible. The molecular weight of this compound should be 240, which is 14 units fewer than infractine. It was temporarily proposed that this compound is pre-infractine (β-carboline-1-propionic acid); the structure is shown in Figure 2, compound b.


Figure 2. Structures of indole alkaloids

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Conclusions

SFC-MS equipped with the Discovery HS F5 column was proven to be a powerful tool in separating synthetic alkaloid-like substances (3 , 4) , and likewise it was a valuable tool in this separation of natural alkaloids. Analysis of the indole alkaloid content of C. infractus and C. subtortus showed that C. infractus contained preinfractine (β-carboline-1-propionic acid) and infractopicrine. However, neither compound was found in C. subtortus. The chemotaxonomic evidence does not support a close relationship between C. infractus and C. subtortus. This research supports the separation of these two species into two different sections, Infracti and Subtorti, following the previous proposal by Brandrud et al. (5) .

Taking into consideration the molecular analysis by Garnica et al. (1) and a SFC-MS analysis conducted during this study, it is accepted that these two sections belong to the same monophyletic clade.

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Materials

     

References

  1. Garnica, S., Wei, M., Oertel, B. & Oberwinkler, F. 2003. Phylogenetic relationships of European Phlegmacium species (Cortinarius, Agaricales). Mycologia 95: 1155-1170.
  2. Steglich, W., Kopanski, L., Wolf, M., Moser, M. & Tegtmeyer, G. 1984. Indolalkaloide aus dem Bltterpilz Cortinarius infractus (Agaricales). Tetrahedron Letters 25: 2341-2344.
  3. Brondz, I., Klein U., Karaliova, L., Jennings, I., Hustad, J. A. & Svendsen R. 2006. Separation of the Positional Isomer Quinocide from the Anti-malaria Drug Primaquine Using a Discovery HS F5 HPLC Column. Trends in Chromatography, in press.
  4. Brondz, I., Hoiland, K., Bell, D., Annino, A., Langley, J., Mohini, T., Amaury, C. & Oakly, P. 2006. Chemotaxonomic comparison of Cortinarius infractus and Cortinarius subtortus using SFC fingerprint and SFC-MS analysis of indole alkaloids content. Mycological Research, in press.
  5. Brandrud, T.E., Lindström, H., Marklund, H., Melot, J. & Muskos, S. 1989-1998. Cortinarius Flora Photographica I-IV. Cortinarius HB, Matfors.

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