A new analytical method for high-sensitivity direct detection, quantitation, and isotopomer analysis of antimalarial drug artemisinin (qinghaosu) is described using HPLC−electrospray ionization mass spectrometry, without pre- or postcolumn derivatization. The method has been shown to be particularly suitable for analysis of artemisinin in biosynthetic stable isotope labeling experiments and quantitative analysis in the nanogram range in crude biological samples such as Artemisia annua plant extracts. The method enabled isotopomer analysis in biosynthetic 18O-labeling experiments with plant cell-free enzyme preparation.

To investigate the biosynthetic pathway of artemisinin, an assay system for the determination of activity of the enzymes involved in its synthesis has been developed. Results from these experiments have shown that HEPES provides a better buffer system than Tris-HCl. The enzyme(s) requires Mg2+ and/or Mn2+, and the addition of ATP and NADPH+H+ significantly enhances the enzyme activity. A new substrate, dihydroarteannuin B, has been synthesized that can easily be radiolabeled with high specific activity. It is utilized by the enzyme system and is converted to artemisinin with the same efficiency as the natural substrates. This can be conveniently used as a precursor for elucidation of the pathway for artemisinin biosynthesis.

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