Recent developments in the rapid analysis of plants and tracking their bioactive constituents |
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Authors: | Teris A van Beek Kishore K R Tetala Irina I Koleva Airidas Dapkevicius Vassiliki Exarchou Suzanne M F Jeurissen Frank W Claassen Elbert J C van der Klift |
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Institution: | 1. Natural Products Chemistry Group, Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB, Wageningen, The Netherlands 2. Department of Analytical Chemistry, University of Food Technology, 26 Maritza blv, 4000, Plovdiv, Bulgaria 3. Azorean Centre for Agricultural Technology and Research (CITA-A), Department of Agricultural Science, University of the Azores, Terra Ch?, 9701-851, Angra do Heroísmo, Portugal 4. Laboratory of Analytical Chemistry, Department of Chemistry, University of Ioannina, 45110, Ioannina, Greece 5. RIVM – National Institute for Public Health and the Environment – Centre for Substances and Integrated Risk Assessment, P.O. Box 1, 3720 BA, Bilthoven, The Netherlands
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Abstract: | Natural products chemistry has witnessed many new developments in the last 5 years like extractions with subcritical water
and ionic liquids, LC/HRMS and LC/SPE/cryo-NMR, UHPLC, TLC/MS, MS-based preparative HPLC, comprehensive chromatography (GC × GC,
LC × LC), high-throughput screening, introduction of monolithic columns, miniaturisation, and automated structure identification.
Nevertheless identifying bioactive constituents in complex plant extracts remains a tedious process. The classical approach
of bioassay guided fractionation is time-consuming while off-line screening of extracts does not provide information on individual
compounds and sometimes suffers from false positives or negatives. One way out of this is by coupling chromatography with
chemical or biochemical assays, so called high resolution screening. An example is the development of HPLC on-line assays
for antioxidants. By the post-column addition of a relatively stable coloured radical like DPPH• or ABTS•+, radical scavengers are detected as negative peaks because in a reaction coil they reduce the model radical to its reduced,
non-coloured form. When combined with LC/DAD/MS and LC/SPE/NMR, reliable identification of active constituents becomes possible
without the necessity of ever isolating them in a classical sense. Also for finding leads for new drugs, combining HPLC with
biochemical assays is interesting but technically more difficult. Most enzymes do not work at the organic modifier concentrations
commonly encountered in RP-HPLC and the reaction time is often longer requiring dilution and lengthy coils respectively. Therefore,
new techniques have to be implemented to gain the required sensitivity for on-line enzyme assays. For stable analytes, high
temperature LC offers a solution to the organic modifier problem. When enzymes are highly expensive, like those used in the
screening for Cytochrome P450 inhibitors, miniaturisation to chip format may offer a way out. Microreactors (chips) are not
only useful for miniaturising larger assays but also offer completely new prospects in phytochemical analysis. One such application
is in the sample clean-up of acids and bases like alkaloids. In a lay-out of three parallel channels of 100 μm width with
the middle one containing organic phase and the two outer ones water of high pH (feed phase) and low pH (trapping phase) such
a chip replaces two classical LLE steps but is much faster and requires less solvents and less manpower input. |
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Keywords: | Modern phytochemistry On-line HPLC High resolution screening Radical scavenging Liquid– liquid extraction chip |
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