Ilia Brondz* 1 , Klaus Høiland 2 and Jennifer Lefler 3

1. SPONSORS Supercritical fluid chromatography resolution of secondary metabolites and multi-analysis by mass spectrometry–ultraviolet and corona charged aerosol detection Department of Chemistry, Biotechnology and Food Science University of Life Sciences Ilia Brondz*1, Klaus Høiland2 and Jennifer Lefler3 1 Norwegian University 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 University of Oslo, Department of Biology, P.O. Box 1066 Blindern, 0316 Oslo, Norway3 Thar Technologies, Inc., 730 William Pitt Way Pittsburgh, PA 15238, USA. MATERIALS AND METHODS One gram of dried tissue was taken from basidiocarps of each species and then extracted in 10 ml of absolute EtOH (Arcus, Oslo, Norway) for three days. Solid material was removed from the extracts and then stored at – 20 ºC until analysis. Fingerprint analysis of C. infractus and C. subtortus was performed by supercritical fluid chromatography-corona charged aerosol detector -mass spectrometry detection (SFC-CAD-MS). INTRODUCTION The aim of this work was to study of possible connection of detection systems including corona Charged Aerosol Detector (CAD) to Supercritical Fluid Chromatography (SFC) Fig.1. Corona CAD can detect the substances without of UV chromophores and give the total picture about effluent from column it is especially valuable as a nearly universal detector. SFC-corona CAD combined with UV detection can, therefore, recognize eluting substances with and without chromophores present. Additionally, a mass spectrometer can serve as a tool for identification of molecules in the effluent. Secondary metabolites of Cortinarius infractus and C. subtortus were analysed by using SFC equipped with multiple detectors system. In a “fingerprint analysis” performed by SFC equipped with a corona CAD, C. infractus showed some common peaks with C. subtortus Fig. 2. A study comparing the chemical composition of indole alkaloids in the basidiocarps of C. infractus and C. subtortus by SFC and supercritical fluid chromatography – mass spectrometry (SFC-MS) was performed. Cortinarius infractus was found to contain an alkaloid substance of the infractine group, i.e. pre-infractine (β-carboline-1-propionic acid) and infractopicrine. However, no such substances were found in C. subtortus Fig. 3. DISCUSSION In chemotaxonomy, fingerprint chromatography is often used as the first approach towards identification. In some cases it can provide important information, in other cases it can provide important hints for further studies. Fig. 2 line A and B show chromatograms of EtOH extracts of C. infractus and C. subtorus, respectively by using corona CAD detection. Using UV detection in addition to corona CAD facilitated identifying the presence of aromatic substances in the chromatogram, Fig 3. The detector range was further expanded bycombining SFC-UV-CAD with mass spectrometry for the chemotaxonomic analysis of peaks, as demonstrated in Fig 4 and publication [1]. A Micromass PLCZ 4190 mass spectrometer was equipped with ESI running under MassLynx 4.0 (Waters-Micromass, Manchester, UK). They were configured as shown in Fig. 1. A 2-ethyl-pyridine column, 250 mm long, 4.6 mm i.d., 6 mm particle size (Berger Instruments Inc., USA) was used for separation of substances. The flow stream was diverted by a fixed splitters 1/10 and 50/50 and used to feed a MS and corona charged aerosol detector respectively. The parameters for the corona charged aerosol detector were: 5 L/ min N2 (AGA, Oslo, Norway), 2.4 Bar pressure in the detection cell, and current of 100 pA. The MS conditions were: cone voltage of 60 V, extractor voltage 12 V, capillary voltage 4.5 V, ion energy of 1.0 V, multiplier at 400 V, analyser vacuum at 2.6 kPa, desolvation gas flow of 495 L/hr. The mass-to-charge-ratio was scanned in mass range from 50 to 400 automatically. MS was operated in the positive charge mode. UV detection was monitored at 268 nm. Isocratic chromatography was performed using CO2 in the supercritical state with 20 % of the total flow being the modifier MeOH (Merck, Darmstadt, Germany) plus 0.4% diethylamine (Merck). The flow rate was 3 ml/ min and the oven temperature was 35ºC. The back pressure, regulated automatically by back pressure regulator, was 100 Bar. Five microliters (5 mL) of extract were injected by an auto injector. Utilizing Mass Lynx 4.0 allowed researchers to control the MS and the UV parameters and correlate data, however the corona CAD was controlled externally and data from this detector was collected via the CAD native software package. Fig. 3 Infractopicrine Fig. 4 CONCLUSION The combinations of SFC-UV-CAD-MS and SFC-(UV)- CAD-MS were studied. To the best of the knowledge of the authors, the use of the corona (CAD) detection in combination with SFC was not previously described in the literature. In many of publications corona (CAD) detector defined as a universal detector for HPLC. As a universal detector corona (CAD) gives additional power to the use of SFC. Investigation into natural products is difficult because of the limitations of individual detectors. The presented investigation is innovative because it demonstrated the wealth of information that can be harvested by expanding the breath of detection modes when coupled with SFC’s enhanced chromatographic resolving power. Fig. 2 [1] Ilia Brondz, Klaus Høiland, David S. Bell and Amy R. Annino, Indole Alkaloid Separation Using the Discovery HS F5. Chemotaxonomic Study of Two Closely Related Brown - Spored Mushrooms, The Reporter 24. 3 (2006) pp. 6-7.