Method Development Spurs Scientific Progress

New methods have always been a major driving force behind scientific progress. They enabled scientists to venture into new fields of scientific investigations, to tackle issues which could not be addressed before. In the 19^th century, the emergence of cytology as a new discipline would not have been possible without the development of modern optical design by Ernst Abbe. In the 20^th century, the observation of the phenomenon of nuclear magnetic resonance (NMR) by Felix Bloch and Edward M. Purcell paved the way for a new kind of spectroscopic analysis which was soon to become the most powerful method for structural analysis of organic molecules.

New methods may spur paradigm shifts. We are presently witnessing the emergence of a new paradigm in the life sciences. Systems biology would be unthinkable without high-density microarrays, 2D gel electrophoresis, ESI-MS, and bioinformatics. What about natural products research? I would argue that our field is presently undergoing fundamental changes in two major directions: firstly, in the way we can address bioactivity, and secondly in the way we analyze the composition of biological materials and the structures of natural products. Progress in the first direction is closely linked to the advances in systems biology, and to chemoinformatics and development in on-line spectroscopy in the second.

In this issue of Planta Medica, we publish Part 1 of a review on LC-NMR and LC-SPE-NMR, written by Professor Jerzy Jaroszewski, an Advisory Board Member of the journal. It is, to our knowledge, the first article comprehensively reviewing the application of LC-NMR in natural products analysis.

The introduction of robust and sensitive LC-SPE-NMR equipment has been the latest step in a long series of developments in LC-based on-line spectroscopy. It is a breakthrough in the sense that a comprehensive NMR structural analysis of individual compounds in an extract has become feasible at the HPLC scale. In conjunction with other on-line detectors, structure elucidation with just the effluents of an analytical HPLC separation has become reality. Preparative isolation of mg quantities of a compound for the sake of structure elucidation will soon be history. It thus seems an appropriate moment to briefly look back and recall the emergence of LC-based on-line spectroscopy and its impact in natural products analysis.

A computer-controlled photodiode array (PDA) detector for liquid chromatography (LC) was reported in 1976 [1], but it took a few more years until the first commercial photodiode array detector became available in the early 1980s. The potential of the PDA in the field of natural products analysis was rapidly recognized. Notable early examples included the targeted search for new secondary metabolites [2], and the identification of phenolic compounds by the concomitant use of on-line UV-vis shift reagents enhancing the spectral information content [3].

The idea of interfacing mass spectrometry with liquid chromatography had been around ever since the successful implementation of GC-MS. Due to the inherent incompatibilities of a liquid mobile phase and a high vacuum mass analyzer, LC-MS remained for a long time the rather exotic playground for a few true believers. Who remembers the days of the moving belt, particle beam, thermospray or continuous flow FAB interfaces? The breakthrough in interface design came with the introduction of atmospheric pressure ionization (API). Ions were now generated outside of the high vacuum part of the mass spectrometer. The atmospheric pressure chemical ionization (APCI) interface was first reported in 1983 [4], soon to be followed by the electrospray (ESI) interface in 1985 [5]. Applications of API-MS in pharmaceutical research followed [6]. The potential of API-MS for the dereplication of natural products in a high-throughput screening (HTS) environment was systematically studied and implemented [7]. Today, LC-MS is a powerful routine tool in numerous natural products laboratories across the world.

Natural products frequently contain stereocenters which can be studied by chiroptical methods such as circular dichroism (CD). Chiroptical stereoanalysis of individual compounds in an extract became feasible with the introduction of an LC-CD detector [8].

Nuclear magnetic resonance (NMR) spectroscopy arguably provides datasets with the highest information content for the analysis of small organic molecules. Again, the idea of using NMR as a LC detection device was put forward long ago. The inherently low sensitivity of NMR and the state of probe design hampered the practical implementation. A first generation of sensitive NMR flow-probes was developed in the early 1990s [9]. First applications of LC-NMR to natural products analysis were soon to follow [10], and the potential of the concerted use of on-line spectroscopy involving LC-NMR was described [11].

Modern LC-SPE-NMR had its conceptual predecessors in the late 1980s [12], but these early approaches were of limited use due to the low sensitivity of the NMR probes of that time. The concept was revived with the advent of newer NMR flow cells [13], [14]. The current state-of-the-art is in cryogenically cooled flow probes and peak collection with a sophisticated SPE interface [15]. These latest developments, and practical applications in the field of natural products will be discussed in Part 2 of the review.

References

• 1 Dessy R E, Reynolds W R, Nunn W G, Titus C A. New mini-computer automated linear photodiode arrays spectrometer system for high-resolution liquid chromatography.  J Chromatogr. 1976;  126 347-68
• 2 Fiedler H P. Screening for new microbial products by high-performance liquid chromatography using a photodiode array detector.  J Chromatogr. 1984;  316 487-94
• 3 Hostettmann K, Domon B, Schaufelberger D, Hostettmann M. On-line high-performance liquid chromatography - Ultraviolet-visible spectroscopy of phenolic compounds in plant extracts using post-column derivatization.  J Chromatogr. 1984;  283 137-47
• 4 Thomson B A, Danylewychmay L, Henion J D. LC-MS-MS with an atmospheric pressure chemical ionization source. Abstr Pap Amer Chem Soc 1983: 19
• 5 Whitehouse C M, Dreyer R N, Yamashita M, Fenn J B. Electrospray interface for liquid chromatographs and mass spectrometers.  Anal Chem. 1985;  57 675-79
• 6 Fouda H, Nocerini M, Schneider R, Gedutis C. Quantitative analysis by high-performance liquid chromatography atmospheric pressure chemical ionization mass spectrometry - the determination of the renin inhibitor CP-80,794 in human serum.  J Amer Soc Mass Spectrom. 1991;  2 164-7
• 7 Zhou S L, Hamburger M. Application of liquid chromatography atmospheric pressure ionization mass spectrometry in natural products analysis - Evaluation and optimization of electrospray and heated nebulizer interfaces.  J Chromatogr A. 1996;  755 189-204
• 8 Bringmann G, Messer K, Saeb W, Petes E M, Peters K. The absolute configuration of (1)-isoshinanolone and in situ LC-CD analysis of its stereoisomers from crude extracts.  Phytochemistry. 2001;  56 387-91
• 9 Spraul M, Hofmann M, Dvortsak P, Nicolson J K, Wilson I D. High-performance liquid chromatography coupled to high-field proton nuclear magnetic resonance spectroscopy: application to the urinary metabolites of ibuprofen.  Anal Chem. 1993;  65 327-30
• 10 Spring O, Buschmann H, Vogler B, Schilling E E, Spraul M, Hoffmann M. Sesquiterpene lactone chemistry of Zaluzania grayana from on-line LC-NMR measurements.  Phytochemistry. 1995;  39 609-12
• 11 Hostettmann K, Wolfender J L, Rodriguez S. Rapid detection and subsequent isolation of bioactive constituents of crude plant extracts.  Planta Med. 1997;  63 2-10
• 12 Wilcox C D, Phelan R M. The use of solid-phase extraction columns to effect simple off-line LC/MS, LC/NMR, and LC/FTIR.  J Chromatogr Sci. 1986;  24 130-3
• 13 Griffiths L, Horton R. Optimization of LC-NMR III - Increased signal-to-noise ratio through column trapping.  Magn Reson Chem. 1998;  36 104-9
• 14 Nyberg N T, Baumann H, Kenne L. Application of solid-phase extraction coupled to an NMR flow-probe in the analysis of HPLC fractions.  Magn Reson Chem. 2001;  39 236-40
• 15 Corcoran O, Spraul M. LC-NMR-MS in drug discovery.  Drug Discov Today. 2003;  8 624-31